Procoagulant compounds

ABSTRACT

The present disclosure provides protease-activatable procoagulant compounds comprising a procoagulant polypeptide, e.g., a procoagulant peptide and/or clotting factor, and a linker comprising a protease-cleavable substrate (e.g., a synthetic thrombin substrate) and a self-immolative spacer (e.g., p-amino benzyl carbamate). Upon cleavage of the protease-cleavable substrate by a protease (e.g., thrombin), the self-immolative spacer cleaves itself from the procoagulant polypeptide such that the polypeptide is in an underivatized and active form. Also provided are pharmaceutical compositions, methods for treating bleeding disorders using the disclosed compounds, methods of enhancing in vivo efficacy of procoagulant polypeptides, methods of increasing the efficacy of proteolytic cleavage of compounds comprising procoagulant polypeptides, methods of activating procoagulant polypeptides, and methods of releasing a procoagulant polypeptide from a heterologous moiety such as PEG.

RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No.14/406,163, filed Dec. 5, 2014, now U.S. Pat. No. 10,287,564, which is a35 U.S.C. § 371 filing of International Patent Application No.PCT/US2013/044841, filed Jun. 7, 2013, which claims priority to U.S.Provisional Patent Application Ser. No. 61/800,626, filed Mar. 15, 2013,and 61/657,688, filed Jun. 8, 2012, the entire disclosures of which arehereby incorporated herein by reference.

BACKGROUND Field of the Disclosure

The present invention relates to procoagulant compounds useful for thetreatment of bleeding diseases or disorders.

Background

The blood coagulation pathway, in part, involves the formation of anenzymatic complex of Factor VIIIa (FVIIIa) and Factor IXa (FIXa) (Xasecomplex) on the surface of platelets. FIXa is a serine protease withrelatively weak catalytic activity without its cofactor FVIIIa. The Xasecomplex cleaves Factor X (FX) into Factor Xa (FXa), which in turninteracts with Factor Va (FVa) to cleave prothrombin and generatethrombin. Hemophilia A is a bleeding disorder caused by mutations and/ordeletions in the factor VIII (FVIII) gene resulting in a deficiency ofFVIII activity (Peyvandi et al. 2006). Hemophilia B (also known asChristmas disease) is one of the most common inherited bleedingdisorders in the world. It results in decreased in vivo and in vitroblood clotting activity and requires extensive medical monitoringthroughout the life of the affected individual.

Treatment of hemophilia is by replacement therapy targeting restorationof clotting activity. There are plasma-derived and recombinant clottingfactor products available to treat bleeding episodes on-demand or toprevent bleeding episodes from occurring by treating prophylactically.Based on the half-life of these products, treatment regimens requirefrequent intravenous administration. Such frequent administration ispainful and inconvenient. Strategies to extend the half-life of clottingfactors include pegylation (Rostin J, et al., Bioconj. Chem. 2000;11:387-96), glycopegylation (Stennicke H R, et al., Thromb. Haemost.2008; 100:920-8), formulation with pegylated liposomes (Spira J, et al.,Blood 2006; 108:3668-3673, Pan J, et al., Blood 2009; 114:2802-2811) andconjugation with albumin (Schulte S., Thromb. Res. 2008; 122 Suppl4:S14-9). However, modification of coagulation factors and procoagulantpeptides with half-life extending moieties (e.g., PEG) and other similarstrategies to extend their half-lives can lead to compromised activity.In order to rescue their activity, a cleavable linker can be insertedbetween the protein or peptide of interest and its modifier. The chosencleavable linker must be cleaved efficiently and rapidly by a protease,for example, a protease involved in the coagulation cascade. Thrombinbeing the activator of many clotting factors is the most popular choice.However, all known substrate sequences composed of natural amino acids(e.g., LVPR, ALRPR (SEQ ID NO: 7), etc.) are not optimal substrates.Furthermore, covalent binding of the cleavable linker to a coagulationfactors or procoagulant peptide can result in steric hindrances (e.g.,due to the presence of amino acids such as such as proline, isoleucineor arginine C-terminal to the cleavage site) that can prevent anefficient enzymatic cleavage reaction.

BRIEF SUMMARY

The present disclosure provides procoagulant compounds comprising aprotease-cleavable substrate (e.g., a synthetic thrombin substrate) anda self-immolative spacer (e.g., PABC) linked to a procoagulantpolypeptide, e.g., a clotting factor or a procoagulant peptide.Accordingly, in some embodiments, the present disclosure provides Aprocoagulant compound having a formula:(Het2)-(Pep2)-(Het1)-(L)-Zy-Bx-Pep1  (Formula I)wherein,

Het1 is a first heterologous molecule, which is either absent orpresent;

Het2 is a second heterologous molecule, which is either absent orpresent;

L is a linker, which is either absent or present;

Zy is a protease-cleavable substrate;

Bx is a self-immolative spacer;

Pep1 is a polypeptide; and,

Pep2 is a polypeptide, which is either absent or present;

wherein, Pep1 or Pep2 comprises a clotting factor or a fragment thereof,or a synthetic procoagulant peptide.

In some embodiments, the self-immolative spacer in the procoagulantcompound of the invention undergoes 1,4 elimination after the enzymaticcleavage of the protease-cleavable substrate. In some embodiments, theself-immolative spacer in the procoagulant compound of the inventionundergoes 1,6 elimination after the enzymatic cleavage of theprotease-cleavable substrate. In some embodiments, the self-immolativespacer is a p-amino benzyl carbamate (PABC), a p-amino benzyl ether(PABE), or a p-amino benzyl carbonate. In certain embodiments, theself-immolative spacer comprises an aromatic group. In some embodiments,the aromatic group is selected from the group consisting of benzyl,cinnamyl, naphthyl, and biphenyl. In some embodiments, the aromaticgroup is heterocyclic. In other embodiments, the aromatic groupcomprises at least one substituent. In some embodiments, at least onesubstituent is selected from F, Cl, I, Br, OH, methyl, methoxy, NO₂,NH₂, NO³⁺, NHCOCH₃, N(CH₃)₂, NHCOCF₃, alkyl, haloalkyl, C₁-C₈alkylhalide, carboxylate, sulfate, sulfamate, sulfonate, or anycombinations thereof. In other embodiments, at least one C in thearomatic group is substituted with N, O or C—R₁, wherein R₁ isindependently selected from H, F, Cl, I, Br, OH, methyl, methoxy, NO₂,NH₂, NO³⁺, NHCOCH₃, N(CH₃)₂, NHCOCF₃, alkyl, haloalkyl, C₁-C₈alkylhalide, carboxylate, sulfate, sulfamate, and sulfonate.

In some embodiments, the protease-cleavable substrate in theprocoagulant compound of the invention comprises a coagulation cascadeprotease substrate. In some embodiments, the coagulation cascadeprotease is selected from thrombin, thromboplastin, Factor Va, FactorVIIa, Factor VIIIa, Factor IXa, Factor Xa, Factor XIa, Factor XIIa, orany combinations thereof. In other embodiments, the coagulation cascadeprotease substrate is a thrombin substrate. In some embodiments, thethrombin substrate is a synthetic thrombin substrate. In otherembodiments, the synthetic thrombin substrate comprises the sequence ofD-Phe-Pip-Arg. In some embodiments, the thrombin substrate is selectedfrom D-Phe-Pro-Arg, D-Ala-Leu-Val-Pro-Arg (SEQ ID NO: 17),Ala-Leu-Val-Pro-Arg (SEQ ID NO: 17), Leu-Val-Pro-Arg (SEQ ID NO: 18), orAla-Leu-Arg-Pro-Arg (SEQ ID NO: 90).

In some embodiments, the protease-cleavable substrate comprises acleavage site for a protease selected from neprilysin (CALLA or CDlO),thimet oligopeptidase (TOP), leukotriene A4 hydrolase, endothelinconverting enzymes, ste24 protease, neurolysin, mitochondrialintermediate peptidase, interstitial collagenases, collagenases,stromelysins, macrophage elastase, matrilysin, gelatinases, meprins,procollagen C-endopeptidases, procollagen N-endopeptidases, ADAMs andADAMTs metalloproteinases, myelin associated metalloproteinases,enamelysin, tumor necrosis factor α-converting enzyme, insulysin,nardilysin, mitochondrial processing peptidase, magnolysin,dactylysin-like metalloproteases, neutrophil collagenase, matrixmetallopeptidases, membrane-type matrix metalloproteinases, SP2endopeptidase, prostate specific antigen (PSA), plasmin, urokinase,human fibroblast activation protein (FAPα), trypsin, chymotrypsins,caldecrin, pancreatic elastases, pancreatic endopeptidase,enteropeptidase, leukocyte elastase, myeloblasts, chymases, tryptase,granzyme, stratum corneum chymotryptic enzyme, acrosin, kallikreins,complement components and factors, alternative-complement pathway c3/c5convertase, mannose-binding protein-associated serine protease,coagulation factors, thrombin, protein c, u and t-type plasminogenactivator, cathepsin G, hepsin, prostasin, hepatocyte growthfactor-activating endopeptidase, subtilisin/kexin type proproteinconvertases, furin, proprotein convertases, prolyl peptidases,acylaminoacyl peptidase, peptidyl-glycaminase, signal peptidase,n-terminal nucleophile aminohydrolases, 20s proteasome, γ-glutamyltranspeptidase, mitochondrial endopeptidase, mitochondrial endopeptidaseIa, htra2 peptidase, matriptase, site 1 protease, legumain, cathepsins,cysteine cathepsins, calpains, ubiquitin isopeptidase T, caspases,glycosylphosphatidylinositoliprotein transamidase, cancer procoagulant,prohormone thiol protease, γ-Glutamyl hydrolase, bleomycin hydrolase,seprase, cathepsin D, pepsins, chymosyn, gastricsin, renin, yapsinand/or memapsins, Prostate-Specific antigen (PSA), or any combinationsthereof.

In some embodiments, Pep1 and Pep2 are different. In other embodiments,Pep1 and Pep2 are the same. In some embodiments, Pep1 is a clottingfactor or a fragment thereof. In other embodiments, wherein Pep2 is aclotting factor or a fragment thereof. In certain embodiments, both Pep1and Pep2 are clotting factors or fragments thereof.

In some embodiments, Pep1 is a heavy chain of a clotting factor and Pep2is a light chain of the clotting factor. In other embodiments, theclotting factor is selected from FVII, FVIIa, FVIII, FIX, FX, FXa, vWF,or any combinations thereof. In other embodiments, Pep1 is a syntheticprocoagulant peptide. In other embodiments, Pep2 is a syntheticprocoagulant peptide. In some embodiments, both Pep1 and Pep2 aresynthetic procoagulant peptides.

In some embodiments, the linker (L) is a peptide linker. In someembodiments, the peptide linker comprises at least two amino, at leastthree, at least four, at least five, at least 10, at least 20, at least30, at least 40, at least 50, at least 60, at least 70, at least 80, atleast 90, or at least 100 amino acids. In other embodiments, the peptidelinker comprises at least 200, at least 300, at least 400, at least 500,at least 600, at least 700, at least 800, at least 900, or at least1,000 amino acids. In some embodiments, the peptide linker comprises apeptide having the formula [(Gly)_(x)-Ser_(y)]_(z) where x is from 1 to4, y is 0 or 1, and z is from 1 to 50. In some embodiments, the linker(L) comprises a non-peptide linker.

In some embodiments, the linker (L) consists of a non-peptide linker. Inother embodiments, the non-peptide linker is selected from MC, MP, MPEG,SMCC, MBS, SMPT, LC-SPDP, SMPB, or any combinations thereof.

In some embodiments, the heterologous moiety comprises a half-lifeextending moiety. In other embodiments, the half-life extending moietyis a low-complexity polypeptide. In other embodiments, the half-lifeextending moiety comprises albumin, albumin binding polypeptide or fattyacid, Fc, transferrin, PAS, the C-terminal peptide (CTP) of the βsubunit of human chorionic gonadotropin, polyethylene glycol (PEG),hydroxyethyl starch (HES), albumin-binding small molecules, vWF, XTEN,or any combinations thereof. In some embodiments, the half-lifeextending moiety comprises a clearance receptor or fragment thereofwhich blocks binding of the procoagulant compound to a clearancereceptor. In some embodiments, the clearance receptor is LRP1.

In some embodiments, the heterologous moiety comprises a peptide or apolypeptide which enables visualization or localization of theprocoagulant compound or a fragment thereof. In some embodiments, thevisualization or localization is enabled in vitro, in vivo, ex vivo orany combination thereof. In some embodiments, the peptide or thepolypeptide which enables visualization or localization is selected froma biotin acceptor peptide, a lipoic acid acceptor peptide, a fluorescentprotein, a cysteine-containing peptide for ligation of a biarsenical dyeor for conjugating metastable technetium, a peptide for conjugatingeuropium clathrates for fluorescence resonance energy transfer(FRET)-based proximity assays, or any combination thereof.

In some embodiments, the fluorescent protein is selected from GFP, RFP,YFP, EGFP, EYFP, or any combination thereof. In some embodiments, thebiarsenical dye is 4′,5′-bis(1,3,2-dithioarsolan-2-yl)fluorescein(FlAsH). In some embodiments, the biotin acceptor peptide facilitatesconjugation of avidin- and streptavidin-based reagents. In someembodiments, the lipoic acid acceptor peptide facilitates conjugation ofthiol-reactive probes to bound lipoic acid or direct ligation offluorescent lipoic acid analogs.

In some embodiments, the heterologous moiety comprises a non-peptidicactive agent. In some embodiments, the non-peptidic active agent is aprocoagulant molecule. In some embodiments, the non-peptidic activeagent is a small molecule drug. In some embodiments, the heterologousmoiety comprises a targeting or ligand binding moiety. In someembodiments, the heterologous moiety comprises an anchor or scaffoldingmolecule. In some embodiments, the anchor or scaffolding molecule is alipid, a carbohydrate, or a sulfhydryl group.

In some embodiments, the procoagulant compound of the inventioncomprises the formula Het-L-Zy-Bx-Peb1, wherein:

Het is cysteine,

L is a peptide linker,

Zy is a synthetic thrombin substrate,

Bx is the self-immolative spacer, and

Pep1 is procoagulant peptide.

In some embodiments, the peptide linker comprises a GGGG amino acidsequence, the synthetic thrombin substrate comprises the sequenceDPhe-Pip-Arg, the self-immolative spacer is PABC, and the procoagulantpeptide comprises the sequence:

(SEQ ID NO: 1) rRAPGKLTCLASYCWLFWTGIA.         |        |        S--------S

In some embodiments, the procoagulant compound of the inventioncomprises the formula Zy-Bx-Pep1, wherein: Zy is a synthetic thrombinsubstrate, Bx is a self-immolative spacer, and Pep1 is a clottingfactor. In some embodiments, the synthetic thrombin substrate comprisesthe sequence DPhe-Pip-Arg, the self-immolative spacer is PABC, and theclotting factor is Factor Xa or FVIIa.

In some embodiments, the procoagulant activity of the procoagulantcompounds of the invention is measured using a method selected from anactivated partial thromboplastin time (aPTT) assay, a modified activatedpartial thromboplastin time (aPTT*) assay, a thrombin generation assay(TGA), and a ROTEM assay.

The present disclosure also provides a pharmaceutical compositioncomprising a procoagulant compound of the invention, and apharmaceutically acceptable carrier.

Also provided is a method for treating, ameliorating, or preventing ableeding disease or disorder in a subject, comprising administering tothe subject an effective amount of a procoagulant compound of theinvention or a pharmaceutical composition comprising the procoagulantcompound of the invention. In some embodiments, the bleeding disease ordisorder is caused by a blood coagulation disorder. In some embodiments,the blood coagulation disorder is selected from hemophilia and vonWillebrand disease (vWD). In some embodiments, the blood coagulationdisorder is hemophilia A or hemophilia B. In some embodiments, thebleeding disease or disorder is selected from hemarthrosis, musclebleed, oral bleed, hemorrhage, hemorrhage into muscles, oral hemorrhage,trauma, trauma capitis, gastrointestinal bleeding, intracranialhemorrhage, intra-abdominal hemorrhage, intrathoracic hemorrhage, bonefracture, central nervous system bleeding, bleeding in theretropharyngeal space, bleeding in the retroperitoneal space, andbleeding in the illiopsoas sheath.

The present disclosure also provides a method of treating, ameliorating,or preventing a deficiency in at least one blood coagulation factor inmammalian subject, wherein the blood coagulation factor is selected fromFV, FVII, FVIIa, FVIII, FIX, FX, FXI, and vWF, the method comprisingadministering to the subject an effective amount of the procoagulantcompound of the invention or a pharmaceutical composition comprising theprocoagulant compound. In some embodiments, the subject is a humansubject.

In some embodiments, the procoagulant compound of the invention or apharmaceutical formulation comprising the procoagulant compound of theinvention are used for treating a subject having a blood coagulationdisorder. In some embodiments, the subject is a human subject. In someembodiments, the procoagulant compound of the invention or apharmaceutical formulation comprising the procoagulant compound of theinvention are used for the manufacture of a medicament for thetreatment, prevention, or amelioration of a blood coagulation disorder.

Also provided in the present disclosure is a method for making theprocoagulant compound of the invention comprising using solid-phasepeptide synthesis. In some embodiments, the method uses orthogonalsolid-phase peptide synthesis.

The present disclosure also provides a method of enhancing in vivoefficacy of a procoagulant polypeptide comprising coupling thepolypeptide to a self-immolative spacer, wherein said self-immolativespacer is coupled to a protease-cleavable substrate moiety. In someembodiments, the self-immolative spacer comprises a PABC group. In someembodiments, the protease-cleavable substrate moiety comprises asynthetic thrombin substrate. In some embodiments, the procoagulantpolypeptide is a clotting factor or a procoagulant peptide. In someembodiments, the procoagulant peptide is synthetic.

The present disclosure also provides a method of increasing the efficacyof the cleavage of a protease substrate operably linked to aprocoagulant peptide or clotting factor comprising conjugating aself-immolative linker to said procoagulant polypeptide, wherein saidself-immolative linker is interposed between the protease substrate andthe procoagulant peptide or clotting factor. Also disclosed is a methodof activating a procoagulant peptide comprising contacting aprocoagulant compound of the invention with a protease specific for theprotease-cleavable substrate moiety in said procoagulant compound,wherein the activated procoagulant peptide is released upon proteolyticcleavage of the protease-cleavable substrate moiety.

Also provided is a method of activating a clotting factor comprisingcontacting a procoagulant compound of the invention with a proteasespecific for the protease-cleavable substrate moiety in saidprocoagulant compound, wherein the activated clotting factor is releasedupon proteolytic cleavage of the protease-cleavable substrate moiety.The instant disclosure also provides a method of releasing aprocoagulant peptide from a heterologous moiety comprising contacting aprocoagulant compound of the invention with a protease specific for theprotease-cleavable substrate in said procoagulant compound, wherein theactivated procoagulant polypeptide is released upon proteolytic cleavageof the protease-cleavable substrate.

The present disclosure also provides a method of releasing a clottingfactor from a heterologous moiety comprising contacting a procoagulantcompound of the invention with a protease specific for theprotease-cleavable substrate in said procoagulant compound, wherein theactivated clotting factor is released upon proteolytic cleavage of theprotease-cleavable substrate. In some embodiments, the procoagulantcompound is cleaved by a protease specific for the protease-cleavablesubstrate moiety at least 2-fold, at least 3-fold, at least 4-fold, atleast 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, atleast 9-fold or at least 10-fold-faster when compared to a referenceprocoagulant compound with the same sequence but without aself-immolative linker. In some embodiments, the procoagulant compoundis cleaved by a protease specific for the protease-cleavable substratemoiety at least 20-fold, at least 30-fold, at least 40-fold, at least50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least90-fold or at least 100-fold-faster when compared to a referenceprocoagulant compound with the same sequence but without aself-immolative linker.

In some embodiments, the procoagulant compound of the inventioncomprises a self-immolative spacer comprising an exosite bindingpeptide.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIG. 1A shows the general organization of a protease-activatableprocoagulant compound of the invention. Het2, Pep2, Het1 and L areindependently optional components. Pep1 and Pep2 are polypeptides, atleast one of which is a clotting factor or a procoagulant peptide. Het1and Het are heterologous moieties. L is a linker. Additional linkers canconnect the different moieties; for example, a linker could be locatedbetween Pep2 and Het1 (as shown in the diagram). Additional proteasecleavable substrate and self-immolative spacer groups can be inserted atthe N-terminus of other moieties such as polypeptides or heterologousmoieties. The diagram shows the optional insertion of such a group atthe N-terminus of Pep2.

FIG. 1B is a representation of an exemplary procoagulant compound of theinvention comprising a protease cleavable substrate (Aa₁Aa₂Aa₃Aa₄), aself-immolative spacer and a protein of interest (POI; e.g., a clottingfactor or procoagulant peptide); illustrating the fragmentation of thecompound and the release of the peptide or protein of interest afterproteolytic cleavage of the cleavable substrate and 1,6 spontaneousfragmentation.

FIG. 2 is a representation of an alternative exemplaryprotease-activatable procoagulant compound of the invention whichcomprises an exosite binding peptide (M). The diagram illustrates therelease of the peptide or protein of interest (POI; e.g., a clottingfactor or procoagulant peptide) and the exosite binding peptide afterproteolytic cleavage of a cleavable substrate (Aa₁Aa₂Aa₃Aa₄) and 1,6spontaneous fragmentation.

FIG. 3 and FIG. 4 show the general synthesis scheme forprotease-activatable procoagulant compound of the invention. Thediagrams correspond to the synthesis of Compound 7. FIG. 3 shows thereactions leading to the synthesis of the compound comprising theprotease cleavable substrate (thrombin substrate) and theself-immolative spacer (PABC). FIG. 4 shows the conjugation of thesubstrate/PABC compound to the synthetic procoagulant peptide and thedeprotection of the resulting product to yield Compound 7.

FIG. 5 presents a schematic representation of the cleavage of Compound 7by thrombin.

FIG. 6 shows the kinetics of the cleavage of Compound 7 by thrombin.

FIG. 7 shows the cleavage of Compound 7 during the course of a TGAassay.

FIG. 8 shows the release kinetics of the peptide IVGGQE (SEQ ID NO: 85),which corresponds to the six N-terminal amino acid residues of the heavychain of the FXa clotting factor, from different procoagulant compounds(Compounds 1, 2, and 3) following treatment with 14 nM thrombin.

FIG. 9 shows the release kinetics of the peptide IVGGQE (SEQ ID NO: 85),which corresponds to the six N-terminal amino acid residues of the heavychain of the FXa clotting factor, from different procoagulant compounds(Compounds 1, 4, 5 and 6) following treatment with 1.4 nM thrombin.

FIG. 10 shows the natural processing of factor X to yield activatedfactor X (FXa).

FIG. 11 is a representation of exemplary procoagulant compounds of theinvention comprising FXa clotting factor.

FIG. 12 shows the natural processing of factor VII to yield activatedfactor VII (FVIIa).

FIG. 13 is a representation of exemplary procoagulant compounds of theinvention comprising FVIIa clotting factor.

FIGS. 14A-14 show a flow diagram of a cleavable polypeptide, FVII-186(FIG. 14A) that can be processed by a proprotein convertase (e.g., PACE)to a processed cleavable polypeptide (FIG. 14B). FIG. 14A shows acleavable polypeptide comprising FVIILC (FVII light chain)-ProproteinConvertase Processing Site by a proprotein convertase (e.g., PACEprocessing site, e.g., 2X(RKR) (SEQ ID NO: 88))-Linker1-SUMO-TruncatedFVIIHC (FVII heavy chain without IVGGKV (SEQ ID NO: 83) at theN-terminus)-Linker2-Fc Region2-Linker3-Fc Region2. FIG. 14B shows aschematic diagram of a cleavable polypeptide that has been processed byPACE. The processed cleavable polypeptide comprises two polypeptidechains, the first chain comprising FVIILC linked to the ProproteinConvertase processing site and the second chain comprisingLinker1-SUMO-Truncated FVIIHC (FVII heavy chain without IVGGKV (SEQ IDNO: 83) at the N-terminus)-Linker2-Fc Region1-Linker3-Fc Region2. FIG.14C demonstrates non-reduced (lane 1) or reduced (lane 2) SDS-PAGE,showing the above constructs and chains. (-) indicates a peptide bond.

FIGS. 15A-15D show a flow diagram of (i) FVII-186 cleavage by a SUMOprotease (FIG. 15B) and (ii) its fusion to a thioester peptide (FIG.14C). FIG. 15A is identical to the construct in FIG. 14B. FIG. 15B showsthat, after FVII-186 is cleaved by a SUMO protease, the resultingcleaved polypeptide construct comprises two chains, the first chaincomprising FVIILC and Proprotein Convertase Site and the second chaincomprising Truncated FVIIHC (FVII heavy chain without IVGGKV (SEQ ID NO:83) at the N-terminus)-Linker2-Fc Region1-Linker3-Fc Region2. The firstchain and the second chain are linked by a disulfide bond. FIG. 15Cshows that after the cleaved polypeptide construct in FIG. 15B isligated with a thioester peptide(Biotin-Pra-GGGG-D-Phe-Pip-Arg-PABC-IVGGKV-COSBn (SEQ ID NO: 79)), theresulting construct comprises two polypeptide chains, the first chaincomprising FVIILC and Proprotein Convertase Processing Site and thesecond chain comprising Thrombin cleavage site-FVIIHC (FVII heavychain)-Linker2-Fc Region1-Linker3-Fc Region2 (TA-FVII-186). FIG. 15Dshows reducing SDS-PAGE indicating the constructs and chains: lane 1shows marker; lane 2 shows FVII-186; lane 3 shows FVII-186 with SUMOprotease reaction; lane 3 shows FVII-186 with SUMO protease reaction andconjugation with a positive control peptide; and lane 5 shows FVII-186with SUMO protease reaction and conjugation with PABC peptide. (-)indicates a peptide bond.

FIG. 16 shows FVIIa chromogenic assay after thrombin activation ofTA-FVII-186. X axis indicates time (min), and Y axis indicatesAbsorbance (A405) measurement for FVIIa activity. (x) shows FVIIaactivity of a mixture of thrombin and hirudin. (□) indicates FVIIaactivity of a mixture of FVII-186, thrombin, and hirudin. (∘) indicatesFVIIa activity of a mixture of TA-FVII-186, thrombin, and hirudin.

FIGS. 17A-17 show a flow diagram of FX-011 expression by a proproteinconvertease (e.g., PACE). FIG. 17A shows a cleavable polypeptideconstruct comprising FXLC (Factor X light chain)-AP (activationpeptide)-Proprotein Convertase Processing Site1 (e.g., 2X(RKR) SEQ IDNO: 88))-Truncated FXHC (Factor X heavy chain without six amino acids(i.e., IVGGQE (SEQ ID NO: 85)) at the N-terminus)-Fc Region1-ProproteinConvertase Processing Site2 (e.g., RRRR (SEQ ID NO: 89))-Linker-FcRegion2. When the cleavable construct of FIG. 16A is expressed, theinitial construct can be processed by a proprotein convertase (e.g.,PACE) to three polypeptide chains construct, the first chain comprisingFXLC, the second chain comprising Truncated FXHC (Factor X heavy chainwithout six amino acids (i.e., IVGGQE (SEQ ID NO: 85) at theN-terminus)-Fc Region1-Proprotein Convertase Processing Site2, and thethird chain comprising Fc Region2. FIG. 17C shows reduced (lane 2) andnon-reduced (lane 3) SDS-PAGE showing the chains and constructs. (-)indicates a peptide bond.

FIGS. 18A-18 show a flow diagram of thrombin-cleavable Factor X moleculesynthesis. FIG. 18A construct, identical to FIG. 17B contruct (FX-011),is incubated with a thioester peptide(GG-D-Phe-Pip-Arg-PABC-IVGGQE-COSBn (SEQ ID NO: 80)). The resultingconstruct (TA-FX-011) comprises three chains, the first chain comprisingFXLC, the second chain comprising Thrombin Cleavage Site(D-Phe-Pip-Arg-PABC)-FXHC-Fc Region1-Proprotein Convertase ProcessingSite, and the third chain comprising Fc Region 2. The first chain andthe second chain are bound by a disulfide bond, and the second and thethird chains are bound by two disulfide bonds. FIG. 18C shows theconstructs and chains in SDS-PAGE. (-) indicates a peptide bond.

FIG. 19 shows FXa chromogenic assay after thrombin activation ofTA-FX-011. X axis indicates time (min), and Y axis indicates Absorbance(A405) measurement for FXa activity. (x) shows FXa activity of a mixtureof thrombin and hirudin. (□) indicates FXa activity of a mixture ofFX-011, thrombin, and hirudin. (∘) indicates FXa activity of a mixtureof TA-FXa-011, thrombin, and hirudin.

FIGS. 20A-20C show a flow diagram of a cleavable FX polypeptideconstruct (FX-012). FIG. 20A shows a cleavable polypeptide comprisingFXLC-SUMO-Truncated FXHC (Factor X heavy chain without six amino acids,i.e., IVGGQE (SEQ ID NO: 85) at the N-terminus)-Fc Region1-ProproteinConvertase Processing Site1-Linker-Proprotein Converase ProcessingSite2-Fc Region2. The FIG. 20A construct can be intracellularlyprocessed by a proprotein convertase (e.g., PACE) to result in the FIG.20B construct. FIG. 20B shows a processed cleavable FX polypeptidecomprising three chains (FX-012), the first chain comprising FXLC, thesecond chain comprising SUMO-Truncated FXHC-Fc Region1-ProproteinConvertase Processing Site1, and the third chain comprising Fc Region2.The first chain and the second chain are bound by a disulfide bond. Thesecond chain and the third chain are bound by two disulfide bonds. FIG.20C shows a western blot of FX-012 under non-reduced (Lane 2) andreduced (Lane 3) conditions, compared to a molecular weight marker (Lane1).

FIGS. 21A-21D show a flow diagram of (i) FX-012 cleavage by a SUMOprotease (FIG. 21B) and (ii) its fusion to a thioester peptide (FIG.21C). FIG. 21A is identical to the construct in FIG. 20B. FIG. 21B showsthat, after FX-012 is cleaved by a SUMO protease, the resulting, cleavedpolypeptide construct comprises three polypeptide chains, the firstchain comprising FXLC, the second chain comprising Truncated FXHC(Factor X heavy chain without six amino acids (i.e., IVGGQE (SEQ ID NO:85)) at the N-terminus)-Fc Region1-Proprotein Convertase ProcessingSite2, and the third chain comprising Fc Region2. The first chain andthe second chain are bound by a disulfide bond, and the second and thethird chains are bound by two disulfide bonds. FIG. 21C shows that afterthe cleaved polypeptide construct in FIG. 21B is ligated with athioester peptide (D-Phe-Pip-Arg-PABC-IVGGQE-COSBn (SEQ ID NO: 90)). Theresulting construct (TA-FX-012) comprises three chains, the first chaincomprising FXLC, the second chain comprising Thrombin Cleavage Site(D-Phe-Pip-Arg-PABC)-FXHC-Fc Region1-Proprotein Convertase ProcessingSite, and the third chain comprising Fc Region 2. The first chain andthe second chain are bound by a disulfide bond, and the second and thethird chains are bound by two disulfide bonds. FIG. 21D shows reducingSDS-PAGE indicating the constructs and chains: lane 1 shows FX-012; lane2 shows FX-012 with SUMO protease reaction; lane 3 shows FX-012 withSUMO protease reaction and conjugation with PABC peptide, lane 4 showsFX-012 with SUMO protease reaction and conjugation with a positivecontrol peptide; and lane 5 shows marker. (-) indicates a peptide bond.

FIG. 22 shows FXa chromogenic assay after thrombin activation ofTA-FX-012. X axis indicates time (min), and Y axis indicates Absorbance(A405) measurement for FXa activity. (x) shows FXa activity of a mixtureof thrombin and hirudin. (•) indicates FXa activity of TA-FX-012 withoutthrombin and hirudin. (Δ) indicates FXa activity of a mixture ofTA-FXa-012, thrombin, and hirudin.

DETAILED DESCRIPTION

The present disclosure provides procoagulant compounds comprising aprotease-cleavable substrate (e.g., a synthetic thrombin substrate) anda self-immolative spacer (e.g., PABC) linked to a procoagulantpolypeptide, e.g., a clotting factor or a procoagulant peptide. Theprotease-cleavable substrate can incorporate, e.g., the best knownthrombin substrate, D-PhePipArg. Upon cleavage of the protease-cleavablesubstrate by a protease such as thrombin, the self-immolative spacerallows the release of the polypeptide via spontaneous fragmentation.

I. Definitions

It must be noted that, as used in this specification and the appendedclaims, the singular forms “a”, “an” and “the” include plural referentsunless the context clearly dictates otherwise. The terms “a” (or “an”),as well as the terms “one or more,” and “at least one” can be usedinterchangeably herein.

Furthermore, “and/or” where used herein is to be taken as specificdisclosure of each of the two specified features or components with orwithout the other. Thus, the term “and/or” as used in a phrase such as“A and/or B” herein is intended to include “A and B,” “A or B,” “A”(alone), and “B” (alone). Likewise, the term “and/or” as used in aphrase such as “A, B, and/or C” is intended to encompass each of thefollowing embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C;A and C; A and B; B and C; A (alone); B (alone); and C (alone).

It is understood that wherever embodiments are described herein with thelanguage “comprising,” otherwise analogous embodiments described interms of “consisting of” and/or “consisting essentially of” are alsoprovided.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure is related. For example, the ConciseDictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed.,2002, RC Press; The Dictionary of Cell and Molecular Biology, 3rd ed.,1999, Academic Press; and the Oxford Dictionary Of Biochemistry AndMolecular Biology, Revised, 2000, Oxford University Press, provide oneof skill with a general dictionary of many of the terms used in thisdisclosure.

Units, prefixes, and symbols are denoted in their Système Internationalde Unites (SI) accepted form. Numeric ranges are inclusive of thenumbers defining the range. Unless otherwise indicated, amino acidsequences are written left to right in amino to carboxy orientation. Theheadings provided herein are not limitations of the various embodimentsof the disclosure, which can be had by reference to the specification asa whole. Accordingly, the terms defined immediately below are more fullydefined by reference to the specification in its entirety. Amino acidsare referred to herein by either their commonly known three lettersymbols or by the one-letter symbols recommended by the IUPAC-IUBBiochemical Nomenclature Commission. Nucleotides, likewise, are referredto by their commonly accepted single-letter codes.

The term “sequence” as used to refer to a protein sequence, a peptidesequence, a polypeptide sequence, or an amino acid sequence means alinear representation of the amino acid constituents in the polypeptidein an amino-terminal to carboxyl-terminal direction in which residuesthat neighbor each other in the representation are contiguous in theprimary structure of the polypeptide.

By a “protein” or “polypeptide” is meant any sequence of two or moreamino acids linearly linked by amide bonds (peptide bonds) regardless oflength, post-translation modification, or function. As used herein, theterm “polypeptide” is intended to encompass a singular “polypeptide” aswell as plural “polypeptides.” “Polypeptide,” “peptide,” and “protein”are used interchangeably herein. Thus, peptides, dipeptides,tripeptides, or oligopeptides are included within the definition of“polypeptide,” and the term “polypeptide” can be used instead of, orinterchangeably with any of these terms. The term “polypeptide” is alsointended to refer to the products of post-expression modifications ofthe polypeptide, including without limitation glycosylation,acetylation, phosphorylation, amidation, derivatization by knownprotecting/blocking groups, proteolytic cleavage, or modification bynon-naturally occurring amino acids. A polypeptide can be derived from anatural biological source or produced by recombinant technology, but isnot necessarily translated from a designated nucleic acid sequence. Apolypeptide can be generated in any manner, including by chemicalsynthesis. Also included as polypeptides of the present invention arefragments, derivatives, analogs, or variants of the foregoingpolypeptides, and any combination thereof.

The term “fragment” when referring to polypeptides and proteins of thepresent invention include any polypeptides or proteins which retain atleast some of the properties of the reference polypeptide or protein.E.g., in the case of procoagulant polypeptides such as clotting factorsand procoagulant peptides, a term fragment would refer to anypolypeptides or proteins which retain at least some of the procoagulantactivity of the reference polypeptide or protein. Fragments ofpolypeptides include proteolytic fragments, as well as deletionfragments.

The term “variant” as used herein refers to a polypeptide sequence thatdiffers from that of a parent polypeptide sequence by virtue of at leastone amino acid modification. Variants can occur naturally or benon-naturally occurring. Non-naturally occurring variants can beproduced using art-known mutagenesis techniques. Variant polypeptidescan comprise conservative or non-conservative amino acid substitutions,deletions, or additions.

“Derivatives” of polypeptides or proteins of the invention arepolypeptides or proteins which have been altered so as to exhibitadditional features not found on the native polypeptide or protein. Alsoincluded as “derivatives” are those peptides that contain one or morenaturally occurring amino acid derivatives of the twenty standard aminoacids. A polypeptide or amino acid sequence “derived from” a designatedpolypeptide or protein refers to the origin of the polypeptide.Preferably, the polypeptide or amino acid sequence which is derived froma particular sequence has an amino acid sequence that is essentiallyidentical to that sequence or a portion thereof, wherein the portionconsists of at least 10-20 amino acids, preferably at least 20-30 aminoacids, more preferably at least 30-50 amino acids, or which is otherwiseidentifiable to one of ordinary skill in the art as having its origin inthe sequence.

Polypeptides derived from another peptide can have one or more mutationsrelative to the starting polypeptide, e.g., one or more amino acidresidues which have been substituted with another amino acid residue orwhich has one or more amino acid residue insertions or deletions.Preferably, the polypeptide comprises an amino acid sequence which isnot naturally occurring. Such variants necessarily have less than 100%sequence identity or similarity with the starting antibody. In apreferred embodiment, the variant will have an amino acid sequence fromabout 75% to less than 100% amino acid sequence identity or similaritywith the amino acid sequence of the starting polypeptide, morepreferably from about 80% to less than 100%, more preferably from about85% to less than 100%, more preferably from about 90% to less than 100%(e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) and most preferablyfrom about 95% to less than 100%, e.g., over the length of the variantmolecule. In one embodiment, there is one amino acid difference betweena starting polypeptide sequence and the sequence derived therefrom.Identity or similarity with respect to this sequence is defined hereinas the percentage of amino acid residues in the candidate sequence thatare identical (i.e. same residue) with the starting amino acid residues,after aligning the sequences and intro-ducing gaps, if necessary, toachieve the maximum percent sequence identity.

A polypeptide which is “isolated” is a polypeptide which is in a formnot found in nature. Isolated polypeptides include those which have beenpurified to a degree that they are no longer in a form in which they arefound in nature. In some embodiments, a polypeptide which is isolated issubstantially pure.

A “recombinant” polypeptide or protein refers to a polypeptide orprotein produced via recombinant DNA technology. Recombinantly producedpolypeptides and proteins expressed in host cells are consideredisolated for the purpose of the invention, as are native or recombinantpolypeptides which have been separated, fractionated, or partially orsubstantially purified by any suitable technique. The polypeptidesdisclosed herein, e.g., clotting factors or procoagulant peptides, canbe recombinantly produced using methods known in the art. Alternatively,proteins and peptides disclosed herein can be chemically synthesized.

A “conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art, including basic side chains (e.g., Lys, Arg,His), acidic side chains (e.g., Asp, Glu), uncharged polar side chains(e.g., Gly, Asn, Gln, Ser, Thr, Tyr, Cys), nonpolar side chains (e.g.,Ala, Val, Leu, Ile, Pro, Phe, Met, Trp), beta-branched side chains(e.g., Thr, Val, Ile) and aromatic side chains (e.g., Tyr, Phe, Trp,His). Thus, if an amino acid in a polypeptide is replaced with anotheramino acid from the same side chain family, the substitution isconsidered to be conservative. In another embodiment, a string of aminoacids can be conservatively replaced with a structurally similar stringthat differs in order and/or composition of side chain family members.

Non-conservative substitutions include those in which (i) a residuehaving an electropositive side chain (e.g., Arg, His or Lys) issubstituted for, or by, an electronegative residue (e.g., Glu or Asp),(ii) a hydrophilic residue (e.g., Ser or Thr) is substituted for, or by,a hydrophobic residue (e.g., Ala, Leu, He, Phe or Val), (iii) a cysteineor proline is substituted for, or by, any other residue, or (iv) aresidue having a bulky hydrophobic or aromatic side chain (e.g., Val,He, Phe or Trp) is substituted for, or by, one having a smaller sidechain (e.g., Ala, Ser) or no side chain (e.g., Gly).

The term “percent sequence identity” between two polynucleotide orpolypeptide sequences refers to the number of identical matchedpositions shared by the sequences over a comparison window, taking intoaccount additions or deletions (i.e., gaps) that must be introduced foroptimal alignment of the two sequences. A matched position is anyposition where an identical nucleotide or amino acid is presented inboth the target and reference sequence. Gaps presented in the targetsequence are not counted since gaps are not nucleotides or amino acids.Likewise, gaps presented in the reference sequence are not counted sincetarget sequence nucleotides or amino acids are counted, not nucleotidesor amino acids from the reference sequence.

The percentage of sequence identity is calculated by determining thenumber of positions at which the identical amino-acid residue or nucleicacid base occurs in both sequences to yield the number of matchedpositions, dividing the number of matched positions by the total numberof positions in the window of comparison and multiplying the result by100 to yield the percentage of sequence identity. The comparison ofsequences and determination of percent sequence identity between twosequences can be accomplished using readily available software both foronline use and for download. Suitable software programs are availablefrom various sources, and for alignment of both protein and nucleotidesequences. One suitable program to determine percent sequence identityis bl2seq, part of the BLAST suite of program available from the U.S.government's National Center for Biotechnology Information BLAST website (blast.ncbi.nlm.nih.gov). Bl2seq performs a comparison between twosequences using either the BLASTN or BLASTP algorithm. BLASTN is used tocompare nucleic acid sequences, while BLASTP is used to compare aminoacid sequences. Other suitable programs are, e.g., Needle, Stretcher,Water, or Matcher, part of the EMBOSS suite of bioinformatics programsand also available from the European Bioinformatics Institute (EBI) atebi.ac.uk/Tools/psa.

Different regions within a single polynucleotide or polypeptide targetsequence that aligns with a polynucleotide or polypeptide referencesequence can each have their own percent sequence identity. It is notedthat the percent sequence identity value is rounded to the nearesttenth. For example, 80.11, 80.12, 80.13, and 80.14 are rounded down to80.1, while 80.15, 80.16, 80.17, 80.18, and 80.19 are rounded up to80.2. It also is noted that the length value will always be an integer.

In certain embodiments, the percentage identity “X” of a first aminoacid sequence to a second sequence amino acid is calculated as 100×(Y/Z), where Y is the number of amino acid residues scored as identicalmatches in the alignment of the first and second sequences (as alignedby visual inspection or a particular sequence alignment program) and Zis the total number of residues in the second sequence. If the length ofa first sequence is longer than the second sequence, the percentidentity of the first sequence to the second sequence will be higherthan the percent identity of the second sequence to the first sequence.

One skilled in the art will appreciate that the generation of a sequencealignment for the calculation of a percent sequence identity is notlimited to binary sequence-sequence comparisons exclusively driven byprimary sequence data. Sequence alignments can be derived from multiplesequence alignments. One suitable program to generate multiple sequencealignments is ClustalW2, available from www.clustal.org. Anothersuitable program is MUSCLE, available from drive5.com/muscle/. ClustalW2and MUSCLE are alternatively available, e.g., from the EBI.

It will also be appreciated that sequence alignments can be generated byintegrating sequence data with data from heterogeneous sources such asstructural data (e.g., crystallographic protein structures), functionaldata (e.g., location of mutations), or phylogenetic data. A suitableprogram that integrates heterogeneous data to generate a multiplesequence alignment is T-Coffee, available at tcoffee.org, andalternatively available, e.g., from the EBI. It will also be appreciatedthat the final alignment used to calculate percent sequence identity canbe curated either automatically or manually.

As used herein, the term “half-life” refers to a biological half-life ofa particular polypeptide (e.g., clotting factor or procoagulant peptide)or procoagulant compound of the invention in vivo. Half-life can berepresented by the time required for half the quantity administered to asubject to be cleared from the circulation and/or other tissues in theanimal. When a clearance curve of a given polypeptide or procoagulantcompound of the invention is constructed as a function of time, thecurve is usually biphasic with a rapid α-phase and longer β-phase. Theα-phase typically represents an equilibration of the administered Fcpolypeptide between the intra- and extra-vascular space and is, in part,determined by the size of the polypeptide. The β-phase typicallyrepresents the catabolism of the polypeptide in the intravascular space.In some embodiments, procoagulant compounds of the invention aremonophasic, and thus do not have an alpha phase, but just the singlebeta phase. Therefore, in certain embodiments, the term half-life asused herein refers to the half-life of the procoagulant compound in theβ-phase. The typical β phase half-life of a human antibody in humans is21 days.

The terms “heterologous” and “heterologous moiety” mean that apolynucleotide, polypeptide, or any other moiety which is derived from adistinct entity from that of the entity to which it is being compared.For instance, a heterologous polypeptide can be synthetic, or derivedfrom a different species, different cell type of an individual, or thesame or different type of cell of distinct individuals. In oneembodiment, a heterologous moiety can be a polypeptide fused to anotherpolypeptide to produce a fusion polypeptide or protein. In anotherembodiment, a heterologous moiety can be a non-polypeptide such as PEGconjugated to a polypeptide or protein.

As used herein, the terms “linked,” “fused”, “fusion,” or “connected”refer to linkage via a peptide bond (e.g., genetic fusion), chemicalconjugation, or other means. For example, one way in which molecules ormoieties can be linked employs polypeptide linkers which link themolecules or moieties via peptide bonds. The terms “genetically fused,”“genetically linked” or “genetic fusion” are used interchangeably andrefer to the co-linear, covalent linkage or attachment of two or moreproteins, polypeptides, or fragments thereof via their individualpeptide backbones, through genetic expression of a single polynucleotidemolecule encoding those proteins, polypeptides, or fragments. Suchgenetic fusion results in the expression of a single contiguous geneticsequence. Preferred genetic fusions are in frame, i.e., two or more openreading frames (ORFs) are fused to form a continuous longer ORF, in amanner that maintains the correct reading frame of the original ORFs.Thus, the resulting recombinant fusion protein is a single polypeptidecontaining two or more protein segments that correspond to polypeptidesencoded by the original ORFs.

As used herein the term “moiety” refers to a component part orconstituent of a procoagulant compound of the invention.

As used herein, the term “targeting moiety” refers to heterologousmoiety which localizes or directs the procoagulant compound of theinvention to a desired site or cell. In one embodiment, the procoagulantcompound of the invention comprises a “targeting moiety” which enhancesthe activity of the procoagulant compound, e.g., by localizing it to adesired site. Such a moiety can be, e.g., an antibody or variant thereof(e.g., and scFv) or a peptide. In another embodiment, the procoagulantcompound of the invention comprises a targeting moiety which can be apolypeptide, a receptor binding portion of a ligand, or a ligand bindingportion of a receptor and binds to the desired target, e.g., on a cellor tissue. In some embodiments, the procoagulant compound of theinvention comprises a targeting moiety which is genetically fused,chemically conjugated, or linked to the construct via a linker or othermoiety. Exemplary targeting moieties are described in more detail below.In one embodiment a targeting moiety for use in a procoagulant compoundof the invention comprises an antibody or antibody variant. The term“antibody variant” or “modified antibody” includes an antibody whichdoes not occur in nature and which has an amino acid sequence or aminoacid side chain chemistry which differs from that of a naturally-derivedantibody by at least one amino acid or amino acid modification asdescribed herein. As used herein, the term “antibody variant” includessynthetic forms of antibodies which are altered such that they are notnaturally occurring, e.g., antibodies that comprise at least two heavychain portions but not two complete heavy chains (such as, domaindeleted antibodies or minibodies); multispecific forms of antibodies(e.g., bispecific, trispecific, etc.) altered to bind to two or moredifferent antigens or to different epitopes on a single antigen); heavychain molecules joined to scFv molecules; single-chain antibodies;diabodies; triabodies; and antibodies with altered effector function andthe like.

As used herein the term “scFv molecule” includes binding molecules whichconsist of one light chain variable domain (VL) or portion thereof, andone heavy chain variable domain (VH) or portion thereof, wherein eachvariable domain (or portion thereof) is derived from the same ordifferent antibodies. scFv molecules preferably comprise an scFv linkerinterposed between the VH domain and the VL domain. ScFv molecules areknown in the art and are described, e.g., in U.S. Pat. No. 5,892,019, Hoet al. 1989. Gene 77:51; Bird et al. 1988 Science 242:423; Pantoliano etal. 1991. Biochemistry 30:10117; Milenic et al. 1991. Cancer Research51:6363; Takkinen et al. 1991. Protein Engineering 4:837.

A “scFv linker” as used herein refers to a moiety interposed between theVL and VH domains of the scFv. scFv linkers preferably maintain the scFvmolecule in a antigen binding conformation. In one embodiment, a scFvlinker comprises or consists of an scFv linker peptide. In certainembodiments, a scFv linker peptide comprises or consists of a gly-serpolypeptide linker. In other embodiments, a scFv linker comprises adisulfide bond.

As used herein, the term “protease-cleavable substrate” refers topeptide sequence comprising a site recognized by a protease enzyme.Certain cleavage sites comprise an intracellular processing site. In oneembodiment, a procoagulant compound of the invention comprises aprotease-cleavable substrate cleaved by an enzyme that is activatedduring the dotting cascade, such that cleavage of such sites occurs atthe site of clot formation. Exemplary protease-cleavable substratesinclude, e.g., those recognized by thrombin, Factor XIa or Factor Xa.Exemplary FXIa cleavage sites include, e.g., TQSFNDFTR (SEQ ID NO: 2)and SVSQTSKLTR (SEQ ID NO: 3). Exemplary thrombin cleavage sitesinclude, e.g., DFLAEGGGVR (SEQ ID NO: 4), TTKIKPR (SEQ ID NO: 5), LVPRG(SEQ ID NO: 6) and ALRPR (SEQ ID NO: 7). Other enzymatic cleavage sitesare known in the art. Protease-cleavable substrates can comprise naturalor non-natural amino acids, e.g., D-amino acids.

The term “bleeding disease or disorder,” as used herein, means agenetically inherited or acquired condition characterized by a tendencyto hemorrhage, either spontaneously or as a result of trauma, due to animpaired ability or inability to form a fibrin clot. Examples of suchdisorders include hemophilias. The three main forms are hemophilia A(factor VIII deficiency), hemophilia B (factor IX deficiency or“Christmas disease”) and hemophilia C (factor XI deficiency, mildbleeding tendency). Other hemostatic disorders include, e.g., vonWillebrand disease, Factor XI deficiency (PTA deficiency), Factor XIIdeficiency, deficiencies or structural abnormalities in fibrinogen,prothrombin, Factor V, Factor VII, Factor X or factor XIII,Bernard-Soulier syndrome, which is a defect or deficiency in GPIb. GPIb,the receptor for vWF, can be defective and lead to lack of primary clotformation (primary hemostasis) and increased bleeding tendency), andthrombasthenia of Glanzman and Naegeli (Glanzmann thrombasthenia). Inliver failure (acute and chronic forms), there is insufficientproduction of coagulation factors by the liver; this can increasebleeding risk.

The phrase “effective amount” as used herein refers to that amount of aprocoagulant compound or pharmaceutical composition of the presentinvention, which is effective for producing a desired effect, at areasonable benefit/risk ratio applicable to any medical treatment. Forexample, an “effective amount” is an amount effective to reduce orlessen at least one symptom of the disease or disorder being treated orto reduce or delay onset of one or more clinical markers or symptomsassociated with the disease or disorder, or to modify or reverse thedisease process.

As used herein, the term “pharmaceutically acceptable” means approved bya regulatory agency of U.S. or E.U. or other government or listed in theU.S. Pharmacopeia or other generally recognized pharmacopeia for use inhumans. Hence, the term “pharmaceutically acceptable” refers to thoseproperties and/or substances that are acceptable to a patient (e.g., ahuman patient) from a toxicological and/or safety point of view.

The term “administering,” as used herein, means to give a procoagulantcompound of the present invention, or pharmaceutical compositioncontaining a procoagulant compound of the present invention, to asubject (e.g., human subject) in need thereof via a pharmaceuticallyacceptable route of administration. In some embodiments, the route ofadministration is intravenous, e.g., intravenous injection orintravenous infusion. In other embodiments, the route of administrationis selected from subcutaneous, intramuscular, oral, nasal, and pulmonaryadministration. The procoagulant compounds of the invention can beadministered as part of a pharmaceutical composition comprising at leastone pharmaceutically acceptable carrier.

The term “prophylactic treatment,” as used herein, means administering aprocoagulant compound of the present invention to a subject over acourse of time to increase the level of activity in a subject's plasma.Preferably, the increased level is sufficient to decrease the incidenceof spontaneous bleeding or to prevent bleeding, e.g., in the event of anunforeseen injury. Preferably, during prophylactic treatment, the plasmaprotein level in the subject does not fall below the baseline level forthat subject, or below the level that characterizes severe hemophilia.

The term “subject,” as used herein means a human or a non-human mammal.Non-human mammals include, e.g., mice, dogs, primates, monkeys, cats,horses, cows, pigs, and other domestic animals and small animals.

The term “therapeutic dose,” as used herein, means a dose that achievesa therapeutic goal, as described herein. The therapeutic doses that canbe used in the methods of the invention are about 10-100 mg/kg, morespecifically, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, or90-100 mg/kg, and more specifically, 10, 15, 20, 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 mg/kg. Additional therapeuticdoses that can be used in the methods of the invention are about 10 toabout 150 mg/kg, more specifically, about 100-110, 110-120, 120-130,130-140, 140-150 mg/kg, and more specifically, about 110, 115, 120, 125,130, 135, 140, 145, or 150 mg/kg.

“About,” as used herein for a range, modifies both ends of the range.Thus, “about 10-20” means “about 10 to about 20.”

The term “selective” as used in connection with proteolytic cleavagemeans a greater rate of cleavage of a protease-cleavable substraterelative to cleavage of a peptide substrate which comprises a randomsequence of amino acids. The term “selective” also indicates that theprocoagulant compound comprising the protease-cleavable substrate iscleaved at the site where it is coupled to the amino group of theself-immolative spacer.

The term “D-amino acid” as used herein, refers to an amino acid having aD-configuration (e.g., D-Phe). A D-amino acid can be a naturallyoccurring amino acid or an unnatural amino acid.

The term “self-immolative spacer” as used herein refers to abifunctional chemical moiety which is capable of covalently linkingtogether two spaced moieties (e.g., a clotting factor or a procoagulantpeptide and a protein-cleavable substrate) into a normally stabletripartate molecule. The self-immolative spacer will spontaneouslyseparate from the second moiety (e.g., a clotting factor or aprocoagulant peptide) if its bond to the first moiety (e.g., aprotein-cleavable substrate) is cleaved.

II. Procoagulant Compounds

The present disclosure provides procoagulant compounds comprising aclotting factor or a fragment, variant, or derivative thereof, or aprocoagulant peptide (e.g., a synthetic procoagulant peptide) connectedto a protease-cleavable substrate (e.g., a thrombin substrate) via aself-immolative spacer (e.g., PABC). The PABC self-immolative spacerallows the release of any peptides and proteins containing at least oneamine, phenol, carboxylic acid, or thiol functionality upon cleavage ofthe protease-cleavable substrate by endogenous or exogenous proteasesand 1,6-spontaneous fragmentation. Cleavage kinetics is independent ofthe identity of released amine, phenol, carboxylic acid, or thiolmolecules. Moreover, PABC can enhance the cleavage rate due to thepresence of the p-aminobenzyl group.

In some embodiments, the disclosure provides a procoagulant compoundrepresented by the following general formula:(Het2)-(Pep2)-(Het1)-(L)-Zy-Bx-Peb1  (Formula I)wherein:

Het1 is a first heterologous molecule, which is either absent orpresent;

Het2 is a second heterologous molecule, which is either absent orpresent;

L is a linker, which is either absent or present;

Zy is a protease-cleavable substrate;

Bx is a self-immolative spacer;

Pep1 is a first polypeptide; and,

Pep2 is a second polypeptide, which is either absent or present;

wherein, Pep1 or Pep2 is a clotting factor or a fragment thereof, or aprocoagulant peptide (for example, a synthetic procoagulant peptide)(see FIG. 1A).

In various embodiments, the present disclosure provides, inter alia,procoagulant compounds which are selectively activatable at the site ofinjury; procoagulant compounds that are selectively activatable byclotting cascade proteases; methods of treatment of bleeding disorderscomprising the administration of the procoagulant compounds of thedisclosure; methods for the production of the procoagulant compounds ofthe disclosure; and pharmaceutical compositions comprising theprocoagulant compounds of the disclosure.

In some embodiments, the procoagulant compounds disclosed herein arestable and pharmacologically inactive in the absence of the proteasetargeting the protease-cleavable substrate. However, upon action of theprotease, or any other suitable cleavage conditions, theprotease-cleavable substrate is cleaved and the self-immolative spacerundergoes a spontaneous reaction, resulting in the release of an activeprocoagulant polypeptide (e.g., an active clotting factor or aprocoagulant peptide). In some embodiments, the procoagulant compound ofthe invention is a zymogen.

In some aspects, a procoagulant protein of the invention comprises aformula selected from:

(a) Zy-Bx-Peb1;

(b) Het1-Zy-Bx-Peb1;

(c) Het1-L-Zy-Bx-Peb1;

(d) Pep2-Zy-Bx-Peb1;

(e) Pep2-L-Zy-Bx-Pep1;

(f) Pep2-Het1-L-Zy-Bx-Peb1;

(g) Pep2-Het1-Zy-Bx-Peb1;

(h) Het2-Het1-L-Zy-Bx-Peb1;

(i) Het2-Het1-Zy-Bx-Peb1;

(j) Het2-Pep2-Het1-L-Zy-Bx-Peb1;

(k) Het2-Pep2-L-Zy-Bx-Pep1;

(l) Het2-Pep2-Het1-Zy-Bx-Peb1; or,

(m) Het2-Pep2-Zy-Bx-Pep1,

wherein:

Het1 is a first heterologous molecule;

Het2 is a second heterologous molecule;

L is a linker;

Zy is a protease-cleavable substrate;

Bx is a self-immolative spacer;

Pep1 is a first polypeptide; and,

Pep2 is a second polypeptide;

wherein, Pep1 or Pep2 is a clotting factor or a fragment thereof, or aprocoagulant peptide (for example, a synthetic procoagulant peptide).

In some embodiments, the formulas described herein can compriseadditional sequences between the two moieties, e.g., linkers. Forexample, linkers can be situated between Het2 and Pep2, or between Pep2and Het1. In some embodiments, additional Zy-Bx groups are present atthe N-terminus of peptides (e.g., Pep1 or Pep2) and/or heterologousmolecules (e.g., Het1 or Het2) to facilitate the clean release of suchpeptides and/or heterologous moieties. Accordingly, in some embodiments,a procoagulant protein of the invention comprises a formula selectedfrom:

(n) Pep2-Zy-Bx-L-Zy-Bx-Pep1;

(o) Pep2-Zy-Bx-Het1-L-Zy-Bx-Pep1;

(p) Pep2-Zy-Bx-Het1-Zy-Bx-Peb1;

(q) Het2-Zy-Bx-Pep2-Zy-Bx-Het1-L-Zy-Bx-Pep1;

(r) Het2-Zy-Bx-Pep2-Zy-Bx-L-Zy-Bx-Pep1;

(s) Het2-Zy-Bx-Pep2-Zy-Bx-Het1-Zy-Bx-Pep1, or,

(t) Het2-Zy-Bx-Pep2-Zy-Bx-Pep1,

wherein:

Het1 is a first heterologous molecule;

Het2 is a second heterologous molecule;

L is a linker;

Zy is a protease-cleavable substrate;

Bx is a self-immolative spacer;

Pep1 is a first polypeptide; and,

Pep2 is a second polypeptide;

wherein, Pep1 or Pep2 is a clotting factor or a fragment thereof, or aprocoagulant peptide (e.g., a synthetic procoagulant peptide).

The orientation of the procoagulant compounds formulas herein is listedfrom N-terminus (left) to C-terminus (right). For example, formulaPep2-Zy-Bx-Pep1 means formula NH₂-Pep2-Zy-Bx-Pep1-COOH. Formulas (a) to(t) shown above are included herein merely as non-limiting examples ofprocoagulant compounds of the present invention. For example, theformula Het2-Pep2-Het1-L-Zy-Bx-Pep1 can further comprise sequences atthe free end of Het2, between Het2 and Pep2, between Pep2 and Het1,between Het1 and Sp, between L and Zy, between Zy and Bx, between Bx andPep1, or at the C-terminus of Pep1. In another embodiment, the hyphen(-) indicates a peptide bond or one or more amino acids.

In some embodiments, the procoagulant compound comprises a clottingfactor or fragment thereof and a heterologous moiety. In someembodiments, the heterologous moiety comprises a half-life extendingmoiety selected, e.g., from the group consisting of an immunoglobulinconstant region or portion thereof (e.g., an Fc region), a PAS sequence,HES, and albumin, fragment, or variant thereof, or an XTEN. In yet otherembodiments, the procoagulant compound comprises a clotting factor orfragment thereof, a second clotting factor or fragment thereof, and aPEG heterologous moiety, wherein the procoagulant compound furthercomprises a heterologous moiety selected from an immunoglobulin constantregion or portion thereof (e.g., an Fc region), a PAS sequence, HES, andalbumin, fragment, or variant thereof, or an XTEN.

In other embodiments, the procoagulant compound comprises a clottingfactor or fragment thereof, a synthetic procoagulant polypeptide, and aheterologous moiety, wherein the procoagulant compound further comprisesa second heterologous moiety selected from an immunoglobulin constantregion or portion thereof (e.g., an Fc region), a PAS sequence, HES, andalbumin, fragment, or variant thereof, XTEN, or any combinationsthereof. In other embodiments, the procoagulant compound comprises twosynthetic procoagulant peptides and a heterologous moiety, wherein theprocoagulant compound further comprises a second heterologous moietyselected from an immunoglobulin constant region or portion thereof(e.g., an Fc region), a PAS sequence, HES, and albumin, fragment, orvariant thereof, XTEN, or any combinations thereof. In yet anotherembodiment, the procoagulant compound comprises a clotting factor orfragment thereof, a clotting factor cofactor (e.g., Factor Va if theclotting factor in Factor X; or Tissue Factor if the clotting factor isFactor VII), and a heterologous moiety, wherein the procoagulantcompound further comprises a second heterologous moiety selected from animmunoglobulin constant region or portion thereof (e.g., an Fc region),a PAS sequence, HES, and albumin, fragment, or variant thereof, XTEN, orany combinations thereof.

In specific aspects of the invention, a procoagulant compound of theinvention comprises the formula wherein Het1-L-Zy-Bx-Pep1, wherein Pep1comprises a procoagulant peptide comprising the sequencerRAPGKLTCLASYCWLFWTGIA, Bx comprises a PABC self-immolative spacer, Zycomprises a thrombin-cleavable substrate comprising the sequenceD-Phe-Pip-Arg, L comprises a linker comprising the sequence GGGG, andHet1 comprises a scaffold heterologous moiety comprising a cysteineamino acid.

In a specific aspect of the invention, a procoagulant compound of theinvention comprises the formula wherein Zy-Bx-Pep1, wherein Pep1comprises a FXa clotting factor, Bx comprises a PABC self-immolativespacer, and Zy comprises a thrombin-cleavable substrate comprising thesequence D-Phe-Pip-Arg.

In a specific aspect of the invention, a procoagulant compound of theinvention comprises the formula wherein Zy-Bx-Pep1, wherein Pep1comprises a FVIIa clotting factor, Bx comprises a PABC self-immolativespacer, and Zy comprises a thrombin-cleavable substrate comprising thesequence D-Phe-Pip-Arg.

In another specific aspect of the invention, a procoagulant compound ofthe invention comprises the formula wherein Pep2-Zy-Bx-Pep1, whereinPep1 comprises a FX clotting factor, Bx comprises a PABC self-immolativespacer, Zy comprises a thrombin-cleavable substrate comprising thesequence D-Phe-Pip-Arg, and Pep2 is an activation peptide, whereincleavage of the thrombin-cleavable substrate causes the release of theactivation peptide and the activation of the clotting factor (see FIG.11). In some embodiments according to such formula, the clotting factoris FIXa and the activation peptide is FVIIIa. In other embodiments, theclotting factor is FVIIa and the activation peptide is Tissue Factor. Insome embodiments, the activation peptide can be a procoagulant peptide.

In some specific aspects of the invention, a procoagulant compound ofthe invention comprises the formula Pep1-Het3-Het2-Bx-Het1, wherein Pep1comprises a clotting factor, Het3 is a scaffold heterologous moiety,Het2 is a heterologous moiety, Bx is a self-immolative spacer, and Het1is a second heterologous moiety. In some embodiments according to suchformula, Pep1 comprises a FVIII clotting factor, Het3 is a cysteine,Het2 comprises an Fc heterologous moiety, Bx comprises a PABCself-immolative spacer, and Het1 comprises an XTEN.

In some specific aspects of the invention, a procoagulant compound ofthe invention comprises the formula Pep1-Het2-Bx-Het1, wherein Pep1comprises a clotting factor, Het2 is a scaffold heterologous moiety, Bxis a self-immolative spacer, and Het1 is a second heterologous moiety.In some embodiments according to such formula, Pep1 comprises a FVIIIclotting factor, Het2 is a cysteine, Bx comprises a PABC self-immolativespacer, and Het) comprises an XTEN.

For a better understand of the procoagulant compounds of the disclosuretheir components will be discussed individually below:

A. Polypeptides (e.g., Pep1, Pep2, . . . , Pep_(n))

1. Clotting Factors and Procoagulant Peptides

The procoagulant compounds the invention comprise at least onepolypeptide moiety (Pep1 or Pep2) which is (i) a clotting factor, or(ii) a procoagulant peptide (e.g., a synthetic procoagulant peptide).

The term “clotting factor,” as used herein encompasses clotting factors(e.g., vWF, FV, FVa, FVII, FVIIa, FVIII, FVIIIa, FIX, FIXa, FX, FXa,FXI, FXIa, FXII, FXIIa, FXIII, or FXIIIa), fragments, variants, analogs,or derivatives thereof, naturally occurring, recombinantly produced, orsynthetically produced which prevent or decrease the duration of ableeding episode in a subject. In other words, it means molecules havingprocoagulant activity. In some embodiments, the procoagulant compound ofthe invention comprises a FVII or activated FVII (FVIIa) clottingfactor. In other embodiments, the procoagulant compound of the inventioncomprises a FVIII or activated FVIII (FVIIIa) clotting factor. In someembodiments, the procoagulant compound of the invention comprises a FIXor activated FIX (FIXa) clotting factor. In other embodiments, theprocoagulant compound of the invention comprises a FX or activated FX(FXa) clotting factor. In some embodiments, the procoagulant compound ofthe invention comprises vWF. The term “procoagulant peptide” as usedherein refers to any peptide that has procoagulant activity. Inparticular, the term refers to peptides that initiate or accelerate theprocess of blood coagulation through the transformation of solublecirculating fibrinogen to an insoluble cross-linked fibrin network.

A “synthetic procoagulant peptide” as used herein refers to aprocoagulant polypeptide that has been produced using solid phasepeptide synthesis.

By “procoagulant activity” is meant the ability to promote thrombingeneration and/or fibrin deposition in a suitable test system. A numberof tests are available to assess the function of the coagulation system:activated partial thromboplastin time (aPTT) test, chromogenic assay,ROTEM assay, prothrombin time (PT) test (also used to determine INR),fibrinogen testing (often by the Clauss method), platelet count,platelet function testing (often by PFA-100), TCT, bleeding time, mixingtest (whether an abnormality corrects if the patient's plasma is mixedwith normal plasma), coagulation factor assays, antiphosholipidantibodies, D-dimer, genetic tests (e.g. factor V Leiden, prothrombinmutation G20210A), dilute Russell's viper venom time (dRVVT),miscellaneous platelet function tests, thromboelastography (TEG orSonoclot), thromboelastometry (TEM®, e.g, ROTEM®), or euglobulin lysistime (ELT).

The aPTT test is a performance indicator measuring the efficacy of boththe “intrinsic” (also referred to the contact activation pathway) andthe common coagulation pathways. This test is commonly used to measureclotting activity of commercially available recombinant clottingfactors, e.g., FVIII or FIX. It is used in conjunction with prothrombintime (PT), which measures the extrinsic pathway.

ROTEM analysis provides information on the whole kinetics ofhaemostasis: clotting time, clot formation, clot stability and lysis.The different parameters in thromboelastometry are dependent on theactivity of the plasmatic coagulation system, platelet function,fibrinolysis, or many factors which influence these interactions. Thisassay can provide a complete view of secondary haemostasis.

In some embodiments, the procoagulant compound comprises a singleclotting factor. In some embodiments, the single clotting factor isPen1. In other embodiments, the single clotting factor is Pep2. In otherembodiments, the procoagulant compound comprises two clotting factors.In some embodiments, the two clotting factors are the same. In otherembodiments, the two clotting factors are different. In someembodiments, one clotting factor is a fragment of a clotting factor(e.g., the heavy chain of a clotting factor such as FVIII) and thesecond clotting factor is a fragment of the same clotting factor (e.g.,the light chain of a clotting factor such as FVIII). In someembodiments, the procoagulant compound comprises more than two clottingfactors.

In some embodiments, a clotting factor's amino terminus is linked to aself-immolative spacer, which in turn is linked to a protease-cleavablesubstrate. In some embodiments, a procoagulant compound of the inventioncomprises two clotting factors (e.g., two different clotting factors orthe heavy and light chains of a clotting factor) wherein only one ofthem has its amino terminus linked to a self-immolative spacer, which inturn is linked to a protease-cleavable substrate. In other embodiments,a procoagulant compound of the invention comprises two clotting factors(e.g., two different clotting factors or the heavy and light chains of aclotting factor) wherein both of them have its amino terminus linked toa self-immolative spacer, which in turn is linked to aprotease-cleavable substrate.

In some embodiments, the procoagulant compound comprises a procoagulantpeptide (e.g., a procoagulant synthetic peptide). In some embodiments,the procoagulant compound comprises two procoagulant peptides. In someembodiments, the two procoagulant peptides can be the same. In otherembodiments, the two procoagulant peptides can be different. In someembodiments, at least one procoagulant peptide is a syntheticprocoagulant peptide. In some embodiments, both procoagulant peptidesare synthetic. In some embodiments, the procoagulant compound comprisesmore than two procoagulant peptides.

In some embodiments, the procoagulant compound comprises a clottingfactor and a procoagulant peptide, e.g. a synthetic procoagulantpeptide. In some embodiments, Pep1 is a clotting factor and Pep2 is aprocoagulant peptide, e.g., a synthetic procoagulant peptide. In otherembodiments, Pep1 is a procoagulant peptide, e.g., a syntheticprocoagulant peptide, and Pep2 is a clotting factor.

In some embodiments, Pep1 and Pep2 are a clotting factor-clottingcofactor pair, e.g., FVIIa and tissue factor, FVIII and FIX, FVIII andvWF, FIXa and FVIIIa, etc.

Suitable clotting factors and procoagulant peptides to incorporate asPep1 and/or Pep2 in procoagulant compounds of the invention aredisclosed for example in WO2011/069164; WO 2012/006624; WO 2004/101740;WO/2007/112005; and WO 2012/006633; and in U.S. Provisional PatentApplications 61/491,762; 61/467,880; 61/442,029; 61/363,186; 61/599,305;61/586,654; 61/586,099; 61/622,789; 61/586,443; 61/569,158; 61/541,561;61/522,647; 61/506,015; 61/496,542; 61/496,541; 61/496,544; 61/496,543;or 3480000; all of which are herein incorporated by reference in theirentireties.

(i) Clotting Factors

In some embodiments, Pep1 and/or Pep2 are clotting factors, e.g., FactorVII, Factor VIII, Factor IX, and Factor X. Active forms of Factors VII,IX, and X are comprised of dimeric molecules in which the heavy andlight chain are linked only by a disulfide bond. Methods for activatingclotting factors are known in the art.

a. Factor VII

In some embodiments, a clotting factor is a mature form of Factor VII ora variant thereof. Factor VII (FVII, F7; also referred to as Factor 7,coagulation factor VII, serum factor VII, serum prothrombin conversionaccelerator, SPCA, proconvertin and eptacog alpha) is a serine proteasethat is part of the coagulation cascade. FVII includes a Gla domain, twoEGF domains (EGF-1 and EGF-2), and a serine protease domain (orpeptidase S1 domain) that is highly conserved among all members of thepeptidase S1 family of serine proteases, such as for example withchymotrypsin. FVII occurs as a single chain zymogen, an activatedzymogen-like two-chain polypeptide and a fully activated two-chain form.

As used herein, a “zymogen-like” protein or polypeptide refers to aprotein that has been activated by proteolytic cleavage, but stillexhibits properties that are associated with a zymogen, such as, forexample, low or no activity, or a conformation that resembles theconformation of the zymogen form of the protein. For example, when it isnot bound to tissue factor, the two-chain activated form of FVII is azymogen-like protein; it retains a conformation similar to the uncleavedFVII zymogen, and, thus, exhibits very low activity. Upon binding totissue factor, the two-chain activated form of FVII undergoesconformational change and acquires its full activity as a coagulationfactor.

Exemplary FVII variants include those with increased specific activity,e.g., mutations that increase the activity of FVII by increasing itsenzymatic activity (Kcat or Km). Such variants have been described inthe art and include, e.g., mutant forms of the molecule as described forexample in Persson et al. 2001. PNAS 98:13583; Petrovan and Ruf. 2001.J. Biol. Chem. 276:6616; Persson et al. 2001 J. Biol. Chem. 276:29195;Soejima et al. 2001. J. Biol. Chem. 276:17229; Soejima et al. 2002. J.Biol. Chem. 247:49027.

In one embodiment, a variant form of FVII includes the mutationsExemplary mutations include V158D-E296V-M298Q. In another embodiment, avariant form of FVII includes a replacement of amino acids 608-619(LQQSRKVGDSPN, corresponding to the 170-loop) from the FVII maturesequence with amino acids EASYPGK from the 170-loop of trypsin. Highspecific activity variants of FIX are also known in the art. Forexample, Simioni et al. (2009 N.E. Journal of Medicine 361:1671)describe an R338L mutation. Chang et al. (1988 JBC 273:12089) and Pierriet al. (2009 Human Gene Therapy 20:479) describe an R338A mutation.Other mutations are known in the art and include those described, e.g.,in Zogg and Brandstetter. 2009 Structure 17:1669; Sichler et al. 2003.J. Biol. Chem. 278:4121; and Sturzebecher et al. 1997. FEBS Lett412:295. The contents of these references are incorporated herein byreference.

Full activation, which occurs upon conformational change from azymogen-like form, occurs upon binding to is co-factor tissue factor.Also, mutations can be introduced that result in the conformation changein the absence of tissue factor. Hence, reference to FVIIa includes bothtwo-chain forms thereof: the zymogen-like form, and the fully activatedtwo-chain form.

b. Factor VIII

In one embodiment, a clotting factor is a mature form of Factor VIII ora variant thereof. FVIII functions in the intrinsic pathway of bloodcoagulation as a cofactor to accelerate the activation of factor X byfactor IXa, a reaction that occurs on a negatively charged phospholipidsurface in the presence of calcium ions. FVIII is synthesized as a 2351amino acid single-chain polypeptide having the domain structureA1-A2-B-A3-C1-C2. Wehar, G. A. et al., Nature 312:337-342 (1984) andToole, J. J. et al., Nature 312:342-347 (1984).

The domain structure of FVIII is identical to that of the homologouscoagulation factor, factor V (FV). Kane, W. H. et al., PNAS (USA)83:6800-6804 (1986) and Jenny, R. J. et al., PNAS (USA) 84:4846-4850(1987). The FVIII A-domains are 330 amino acids and have 40% amino acididentity with each other and to the A-domain of FV and the plasmacopper-binding protein ceruloplasmin. Takahashi, N. et al., PNAS (USA)81:390-394 (1984). Each C-domain is 150 amino acids and exhibits 40%identity to the C-domains of FV, and to proteins that bindglycoconjugates and negatively charged phospholipids. Stubbs, J. D. etal., PNAS (USA) 87:8417-8421 (1990). The FVIII B-domain is encoded by asingle exon and exhibits little homology to any known protein includingFV B-domain. Gitschier, J. et al., Nature 312:326-330 (1984) and Cripe,L. D. et al., Biochemistry 31:3777-3785 (1992).

FVIII is secreted into plasma as a heterodimer of a heavy chain (domainsA1-A2-B) and a light chain (domains A3-C1-C2) associated through adivalent metal ion linkage between the A1- and A3-domains. In plasma,FVIII is stabilized by binding to von Willebrand factor. Morespecifically, the FVIII light chain is bound by noncovalent interactionsto a primary binding site in the amino terminus of von Willebrandfactor.

Upon proteolytic activation by thrombin, FVIII is activated to aheterotrimer of 2 heavy chain fragments (A1, a 50 kDa fragment, and A2,a 43 kDa fragment) and the light chain (A3-C1-C2, a 73 kDa chain). Theactive form of FVIII (FVIIIa) thus consists of an A1-subunit associatedthrough the divalent metal ion linkage to a thrombin-cleaved A3-C1-C2light chain and a free A2 subunit associated with the A1 domain throughan ion association. Eaton, D. et al., Biochemistry 25: 505 (1986);Lollar, P. et al., J. Biol. Chem. 266: 12481 (1991); and Fay, P. J. etal., J. Biol. Chem. 266: 8957 (1991). This FVIIIa heterotrimer isunstable and subject to rapid inactivation through dissociation of theA2 subunit under physiological conditions.

In one embodiment, a clotting factor comprises a B-domain deletedversion of factor VIII. “B-domain” of Factor VIII, as used herein, isthe same as the B-domain known in the art that is defined by internalamino acid sequence identity and sites of proteolytic cleavage, e.g.,residues Ser741-Arg1648 of full-length human Factor VIII. The otherhuman Factor VIII domains are defined by the following amino acidresidues: A1, residues Ala1-Arg372; A2, residues Ser373-Arg740; A3,residues Ser1690-Asn2019; C1, residues Lys2020-Asn2172; 2, residuesSer2173-Tyr2332. The A3-C1-C2 sequence includes residuesSer1690-Tyr2332. The remaining sequence, residues Glu1649-Arg1689, isusually referred to as the a3 acidic region.

The locations of the boundaries for all of the domains, including theB-domains, for porcine, mouse and canine Factor VIII are also known inthe art. In one embodiment, the B domain of Factor VIII is deleted(“B-domain-deleted factor VIII” or “BDD FVIII”). An example of a BDDFVIII is REFACTO® (recombinant BDD FVIII with S743/Q1638 fusion), whichis known in the art.

A “B-domain-deleted Factor VIII” can have the full or partial deletionsdisclosed in U.S. Pat. Nos. 6,316,226, 6,346,513, 7,041,635, 5,789,203,6,060,447, 5,595,886, 6,228,620, 5,972,885, 6,048,720, 5,543,502,5,610,278, 5,171,844, 5,112,950, 4,868,112, and 6,458,563, each of whichis incorporated herein by reference in its entirety. In someembodiments, a B-domain-deleted Factor VIII sequence comprises any oneof the deletions disclosed at col. 4, line 4 to col. 5, line 28 andexamples 1-5 of U.S. Pat. No. 6,316,226 (also in U.S. Pat. No.6,346,513).

In another embodiment, a B-domain deleted Factor VIII is the S743/Q1638B-domain deleted Factor VIII (SQ version Factor VIII) (e.g., Factor VIIIhaving a deletion from amino acid 744 to amino acid 1637, e.g., FactorVIII having amino acids 1-743 and amino acids 1638-2332). In someembodiments, a B-domain-deleted Factor VIII of the present invention hasa deletion disclosed at col. 2, lines 26-51 and examples 5-8 of U.S.Pat. No. 5,789,203 (also U.S. Pat. Nos. 6,060,447, 5,595,886, and6,228,620).

In some embodiments, a B-domain-deleted Factor VIII has a deletiondescribed in col. 1, lines 25 to col. 2, line 40 of U.S. Pat. No.5,972,885; col. 6, lines 1-22 and example 1 of U.S. Pat. No. 6,048,720;col. 2, lines 17-46 of U.S. Pat. No. 5,543,502; col. 4, line 22 to col.5, line 36 of U.S. Pat. No. 5,171,844; col. 2, lines 55-68, FIG. 2, andexample 1 of U.S. Pat. No. 5,112,950; col. 2, line 2 to col. 19, line 21and table 2 of U.S. Pat. No. 4,868,112; col. 2, line 1 to col. 3, line19, col. 3, line 40 to col. 4, line 67, col. 7, line 43 to col. 8, line26, and col. 11, line 5 to col. 13, line 39 of U.S. Pat. No. 7,041,635;or col. 4, lines 25-53, of U.S. Pat. No. 6,458,563.

In some embodiments, a B-domain-deleted Factor VIII has a deletion ofmost of the B domain, but still contains amino-terminal sequences of theB domain that are essential for in vivo proteolytic processing of theprimary translation product into two polypeptide chain, as disclosed inWO 91/09122, which is incorporated herein by reference in its entirety.In some embodiments, a B-domain-deleted Factor VIII is constructed witha deletion of amino acids 747-1638, i.e., virtually a complete deletionof the B domain. Hoeben R. C., et al. J. Biol. Chem. 265 (13): 7318-7323(1990), incorporated herein by reference in its entirety.

A B-domain-deleted Factor VIII can also contain a deletion of aminoacids 771-1666 or amino acids 868-1562 of Factor VIII. Meulien P., etal. Protein Eng. 2(4): 301-6 (1988), incorporated herein by reference inits entirety. Additional B domain deletions that are part of theinvention include: deletion of amino acids 982 through 1562 or 760through 1639 (Toole et al., Proc. Natl. Acad. Sci. U.S.A. (1986) 83,5939-5942)), 797 through 1562 (Eaton, et al. Biochemistry (1986)25:8343-8347)), 741 through 1646 (Kaufman (PCT published application No.WO 87/04187)), 747-1560 (Sarver, et al., DNA (1987) 6:553-564)), 741though 1648 (Pasek (PCT application No. 88/00831)), or 816 through 1598or 741 through 1648 (Lagner (Behring Inst. Mitt. (1988) No 82:16-25, EP295597)), each of which is incorporated herein by reference in itsentirety. Each of the foregoing deletions can be made in any Factor VIIIsequence.

c. Factor IX

In one embodiment, a clotting factor is a mature form of Factor IX or avariant thereof. Factor IX circulates as a 415 amino acid, single chainplasma zymogen (A. Vysotchin et al., J. Biol. Chem. 268, 8436 (1993)).The zymogen of FIX is activated by FXIa or by the tissue factor/FVIIacomplex. Specific cleavages between arginine-alanine 145-146 andarginine-valine 180-181 result in a light chain and a heavy chain linkedby a single disulfide bond between cysteine 132 and cysteine 289 (S.Bajaj et al., Biochemistry 22, 4047 (1983)).

The structural organization of FIX is similar to that of the vitaminK-dependent blood clotting proteins FVII, FX and protein C (B. Furie andB. Furie, supra). The approximately 45 amino acids of the amino terminuscomprise the gamma-carboxyglutamic acid, or Gla, domain. This isfollowed by two epidermal growth factor homology domains (EGF), anactivation peptide and the catalytic “heavy chain” which is a member ofthe serine protease family (A. Vysotchin et al., J. Biol. Chem. 268,8436 (1993); S. Spitzer et al., Biochemical Journal 265, 219 (1990); H.Brandstetter et al., Proc. Natl. Acad Sci. USA 92, 9796 (1995)).

d. Factor X

In one embodiment, a clotting factor is a mature form of Factor X.Factor X is a vitamin-K dependent glycoprotein of a molecular weight of58.5 kDa, which is secreted from liver cells into the plasma as azymogen. Initially factor X is produced as a prepropeptide with a signalpeptide consisting in total of 488 amino acids. The signal peptide iscleaved off by signal peptidase during export into the endoplasmaticreticulum, the propeptide sequence is cleaved off after gammacarboxylation took place at the first 11 glutamic acid residues at theN-terminus of the mature N-terminal chain. A further processing stepoccurs by cleavage between Arg182 and Ser183. This processing step alsoleads concomitantly to the deletion of the tripeptideArg180-Lys181-Arg182. The resulting secreted factor X zymogen consistsof an N-terminal light chain of 139 amino acids (M, 16,200) and aC-terminal heavy chain of 306 amino acids (M, 42,000) which arecovalently linked via a disulfide bridge between Cys172 and Cys342.Further posttranslational processing steps include the β-hydroxylationof Asp103 as well as N- and O-type glycosylation.

It will be understood that in addition to wild type (WT) versions ofthese clotting factors or biologically active portions thereof, thepresent invention can also employ precursor truncated forms thereof thathave activity, allelic variants and species variants, variants encodedby splice variants, and other variants, including polypeptides that haveat least 40%, 45%, 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, 99% or more sequence identity to the mature form of theclotting factor and which retain the ability to promote clot formation.For example, modified FVII polypeptides and variants thereof whichretain at least one activity of a FVII, such as TF binding, factor Xbinding, phospholipid binding, and/or coagulant activity of a FVII canbe employed. By retaining activity, the activity can be altered, such asreduced or increased, as compared to a wild-type clotting factor so longas the level of activity retained is sufficient to yield a detectableeffect.

Exemplary modified polypeptides include, but are not limited to,tissue-specific isoforms and allelic variants thereof, syntheticmolecules prepared by translation of nucleic acids, proteins generatedby chemical synthesis, such as syntheses that include ligation ofshorter polypeptides, through recombinant methods, proteins isolatedfrom human and non-human tissue and cells, chimeric polypeptides andmodified forms thereof. The instant clotting factors can also consist offragments or portions of WT molecules that are of sufficient length orinclude appropriate regions to retain at least one activity (uponactivation if needed) of a full-length mature polypeptide. Exemplaryclotting factor variants are known in the art.

As used herein, the term “Gla domain” refers to the conserved membranebinding motif which is present In vitamin K-dependent proteins, such asas prothrombin, coagulation factors VII, IX and X, proteins C, S, and Z.These proteins require vitamin K for the posttranslational synthesis ofg-carboxyglutamic acid, an amino acid clustered in the N-terminal Gladomain of these proteins. All glutamic residues present in the domainare potential carboxylation sites and many of them are thereforemodified by carboxylation. In the presence of calcium ions, the Gladomain interacts with phospholipid membranes that includephosphatidylserine. The Gla domain also plays a role in binding to theFVIIa cofactor, tissue factor (TF). Complexed with TF, the Gla domain ofFVIIa is loaded with seven Ca2+ ions, projects three hydrophobic sidechains in the direction of the cell membrane for interaction withphospholipids on the cell surface, and has significant contact with theC-terminal domain of TF.

The Gla domain of factor VII comprises the uncommon aminoacid_-carboxyglutamic acid (Gla), which plays a vital role in thebinding of clotting factors to negatively charged phospholipid surfaces.

The GLA domain is responsible for the high-affinity binding of calciumions. It starts at the N-terminal extremity of the mature form ofproteins and ends with a conserved aromatic residue. A conservedGla-x(3)-Gla-x-Cys motif is found in the middle of the domain whichseems to be important for substrate recognition by the carboxylase.

Using stopped-flow fluorescence kinetic measurements in combination withsurface plasmon resonance analysis, the Gla domain has been found to beimportant in the sequence of events whereby the protease domain of FVIIainitiates contact with sTF (Biochemical and Biophysical ResearchCommunications. 2005. 337:1276). In addition, clearance of clottingfactors can be significantly mediated through Gla interactions, e.g., onliver cells and clearance receptors, e.g., EPCR.

In one embodiment, targeted clotting factors are modified to lack a Gladomain. The Gla domain is responsible for mediating clearance ofclotting factors via multiple pathways, such as binding to liver cells,clearance receptors such as EPCR, etc. Thus, eliminating the Gla domainhas beneficial effects on half life of clotting factors. Though Gladomain is also generally required for activity by localizing clottingfactors to sites of coagulation, the inclusion of a platelet targetingdomain moiety targets the Gla deleted clotting factor to platelets. Inone embodiment, a clotting factor comprises a targeting moiety and lacksa Gla domain. For example, in the case of Factor VII, the Gla domain ispresent at the amino terminus of the light chain and consists of aminoacids 1-35. The Gla domains of exemplary clotting factors are indicatedin the accompanying sequence listing. This domain can be removed usingstandard molecular biology techniques, replaced with a targeting domain,and the modified light chain incorporated into a construct of theinvention. In one embodiment, a cleavage site can be introduced intoconstructs lacking a Gla domain to facilitate activation of themolecule. For example, in one embodiment, such a cleavage site can beintroduced between the amino acids that are cleaved when the clottingfactor is activated (e.g., between amino acids 152 and 153 in the caseof Factor VII).

In one embodiment, a cleavage site can be introduced into constructslacking a Gla domain to facilitate activation of the molecule. Forexample, in one embodiment, such a cleavage site can be introducedbetween the amino acids that are cleaved when the clotting factor isactivated (e.g., between amino acids 152 and 153 in the case of FactorVII). Exemplary clotting factors lacking a Gla domain are shown in theaccompanying figures.

Exemplary clotting factors are those of mammalian, e.g., human, origin.

(ii) Procoagulant Peptides

Suitable procoagulant peptides to incorporate as Pep1 and/or Pep2 inprocoagulant compounds of the invention are disclosed, for example, inU.S. Provisional Application Nos. 61/495,818; 61/600,237; and,61/605,540 which are herein incorporated by reference in theirentireties.

Exemplary synthetic procoagulant peptides include, for example:

(SEQ ID NO: 8) KLTCLASYCWLF; (SEQ ID NO: 9) RRAPGKLTCLASYCWLFWTGIA;(SEQ ID NO: 10) RRAPGKLQCLASYCWLFWTGIA; (SEQ ID NO: 11PRIRTVGPGSRSASGKLTCLASYCWLFWTGIA; (SEQ ID NO: 12)SKQGRPISPDRRAAGKLTCLASYCWLFWTGIA; (SEQ ID NO: 13)PRIRTVGPGSRSASGKSTCLASYCWLFWTGIA; (SEQ ID NO: 14)SRIRTVSPGSRSASGKSTCLASYCWLFWTGIA; or (SEQ ID NO: 15)PRSRTVGPGSRSASGKSTCLASYCWLFWTGIA.2. Other Polypeptides

In some embodiments, the procoagulant compound comprises at least onepolypeptide (e.g., Pep1 or Pep2) that is not a clotting factor or asynthetic procoagulant peptide. In some embodiments, the procoagulantcompound comprises a Pep1 or Pep2 polypeptide which is clotting cascadecofactor, e.g., tissue factor, or a derivative, fragment, or variantthereof.

In other embodiments, the procoagulant compound comprises a Pep1 or Pep2polypeptide comprising a ligand binding moiety. In some embodiments,such ligand binding moiety is an antibody or an antigen binding fragmentthereof.

B. Self-Immolative Spacer (Bx)

Procoagulant compounds according to the present disclosure comprise aself-immolative spacer. In some aspects, the self-immolative spacercomprises an aminobenzyl carbamate group, an aminobenzyl ether group, oran aminobenzyl carbonate group. In one aspect, the self-immolativespacer is p-amino benzyl carbamate (PABC).

P-amino benzyl carbamate (PABC) is the most efficient and mostwidespread connector linkage for self-immolative site-specific prodrugactivation (see, e.g., Carl et al. J. Med. Chem. 24:479-480 (1981); WO1981/001145; Rautio et al, Nature Reviews Drug Discovery 7:255-270(2008); Simplicio et al., Molecules 13:519-547 (2008). PABC allows therelease of any amine drugs, peptides, and proteins upon cleavage by aprotease and 1,6 spontaneous fragmentation (see FIG. 1).

In some embodiments, the self-immolative spacer connects a polypeptideof interest (e.g., a clotting factor or fragment thereof, or a syntheticprocoagulant peptide) to a protease-cleavable substrate (e.g., athrombin substrate). In specific aspects, the carbamate group of a PABCself-immolative spacer is connected to the N-terminus of a polypeptideof interest (e.g., a clotting factor or fragment thereof, or a syntheticprocoagulant peptide), and the amino group of the PABC self-immolativespacer is connected to a protease-cleavable substrate (e.g., a thrombinsubstrate).

The aromatic ring of the aminobenzyl group can optionally be substitutedwith one or more (e.g., R₁ and/or R₂) substituents on the aromatic ring,which replace a hydrogen that is otherwise attached to one of the fournon-substituted carbons that form the ring. As used herein, the symbol“R_(x)” (e.g., R₁, R₂, R₃, R₄) is a general abbreviation that representsa substituent group as described herein.

Substituent groups can improve the self-immolative ability of thep-aminobenzyl group (Hay et al., J. Chem Soc., Perkin Trans. 1:2759-2770(1999); see also, Sykes et al. J. Chem. Soc., Perkin Trans. 1:1601-1608(2000)).

The following formula shows the general topology of a p-amino benzylimmolative linker and the relative locations of an exemplaryprotease-cleavable substrate (Aa₁Aa₂Aa₃Aa₄) and a peptide or protein ofinterest (POI), which could be Pep1, Pep2, a polypeptide heterologousmoiety, or linker comprising a peptide linker. The formula indicatespossible locations of R substituent groups (R₁, R₂, R₃).

The substituents, which can be a single atom, e.g., a halogen, or amulti-atom group, e.g., methyl, are selected in order to impact thestability of the aminobenzyl or the decomposition product thereof.Electron withdrawal from the ring can be used to facilitate thespontaneous decomposition of the aminobenzyl group from the drug aftercleavage of the bond between the amino group of the aminobenzyl groupand the adjacent peptide linkage. Exemplary aromatic group R₁, R₂, or R₃substituents include, for example, F, Cl, I, Br, OH, methyl, methoxy,NO₂, NH₂, NO³⁺, NHCOCH₃, N(CH₃)₂, NHCOCF₃, alkyl, haloalkyl, C₁-C₈alkylhalide, carboxylate, sulfate, sulfamate, sulfonate, etc. (see,e.g., U.S. Pat. Nos. 7,091,186 and 7,659,241). The p-aminobenzyl linkercan comprise a heteroatom Z connected to the amino terminus of thepeptide or protein of interest protein. The term heteroatom, as usedherein, includes oxygen (O), nitrogen (N), sulfur (S), silicon (Si),boron (B) and phosphorus (P). In one embodiment, the heteroatoms in Zare O, S or N.

As illustrated below, in some aspects the self-immolative linkercomprises an M group comprising an exosite binding peptide which bindsto the exosite of its respective clotting factor.

Many exosite binding motifs are known in the art. Insertion of anexosite binding motifs can increase the cleavage rate. Upon cleavage,both the exosite binding motif and the peptide or protein of interest(e.g., a clotting factor or fragment thereof, or a procoagulant peptide)are released (see FIG. 2).

In some embodiments, only one of the four non-substituted carbons in thep-aminobenzyl ring is substituted. In some other embodiments, two of thefour non-substituted carbons in the p-aminobenzyl ring are substituted.In other embodiments, three of the four non-substituted carbons in thep-aminobenzyl ring are substituted. In some embodiments, the fournon-substituted carbons in the p-aminobenzyl ring are substituted.

Self-immolative elimination can take place, e.g., via 1,4 elimination,1,6 elimination (e.g., PABC), 1,8 elimination (e.g., p-amino-cinnamylalcohol), β-elimination, cyclisation-elimination (e.g., 4-aminobutanolester and ethylenediamines), cyclization/lactonization,cyclization/lactolization, etc. See, e.g., Singh et al. Curr. Med. Chem.15:1802-1826 (2008); Greenwald et al. J. Med. Chem. 43:475-487 (2000).

In some aspects, the self-immolative spacer can comprise, e.g., ancinnamyl, naphthyl, or biphenyl groups (see, e.g., Blencowe et al.Polym. Chem. 2:773-790 (2011)). In some aspects, the self-immolativespacer comprises a heterocyclic ring (see, e.g., U.S. Pat. Nos.7,375,078; 7,754,681). Numerous homoaromatic (see, e.g., Carl et al. J.Med. Chem. 24:479 (1981); Senter et al. J. Org. Chem. 55:2975 (1990);Taylor et al. J. Org. Chem. 43:1197 (1978); Andrianomenjanahary et al.Bioorg. Med. Chem. Lett. 2:1903 (1992)), and coumarin (see, e.g.,Weinstein et al. Chem. Commun. 46:553 (2010)), furan, thiophene,thiazole, oxazole, isoxazole, pyrrole, pyrazole (see, e.g., Hay et al.J. Med. Chem. 46:5533 (2003)), pyridine (see, e.g., Perry-Feigenbaum etal. Org. Biomol. Chem. 7:4825 (2009)), imidazone (see, e.g., Nailor etal. Bioorg. Med. Chem. Lett. Z:1267 (1999); Hay and Denny, TetrahedronLett. 38:8425 (1997)), and triazole (see, e.g., Bertrand and Gesson, J.Org. Chem. 72:3596 (2007)) based heteroaromatic groups that areself-immolative under both aqueous and physiological conditions areknown in the art. See also, U.S. Pat Nos. 7,691,962; 7,091,186; U.S.Pat. Publ. Nos. US2006/0269480; US2010/0092496; US2010/0145036;US2003/0130189; US2005/0256030).

In some embodiments, a procoagulant compound of the invention comprisesmore than one self-immolative spacer in tandem, e.g., two or more PABCunits. See, e.g., de Groot et al. J. Org. Chem. 66:8815-8830 (2001). Insome embodiments, a procoagulant compound of the invention can comprisea self-immolative spacer (e.g., a p-aminobenzylalcohol or ahemithioaminal derivative of p-carboxybenzaldehyde or glyoxilic acid)linked to a fluorigenic probe (see, e.g., Meyer et al. Org. Biomol.Chem. 8:1777-1780 (2010)).

Where substituent groups in the self-immolative linkers are specified bytheir conventional chemical formulae, written from left to right, theyequally encompass the chemically identical substituents, which wouldresult from writing the structure from right to left. For example,“—CH₂O—” is intended to also recite “—OCH₂—”. Substituent groups inself-immolative, for example, R₁ and/or R₂ substituents in ap-aminobenzyl self-immolative linker as discuss above can include, e.g.,alkyl, alkylene, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio,heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, aryloxy,heteroaryl, etc. When a compound of the present disclosure includes morethan one substituent, then each of the substituents is independentlychosen.

The term “alkyl,” by itself or as part of another substituent, means,unless otherwise stated, a straight or branched chain hydrocarbonradical having the number of carbon atoms designated (e.g., C₁-C₁₀ meansone to ten carbon atoms). Typically, an alkyl group will have from 1 to24 carbon atoms, for example having from 1 to 10 carbon atoms, from 1 to8 carbon atoms or from 1 to 6 carbon atoms. A “lower alkyl” group is analkyl group having from 1 to 4 carbon atoms. The term “alkyl” includesdi- and multivalent radicals. For example, the term “alkyl” includes“alkylene” wherever appropriate, e.g., when the formula indicates thatthe alkyl group is divalent or when substituents are joined to form aring. Examples of alkyl radicals include, but are not limited to,methyl, ethyl, n-propyl, iso-propyl, n-butyl, tert-butyl, iso-butyl,sec-butyl, as well as homologs and isomers of, for example, n-pentyl,n-hexyl, n-heptyl and n-octyl.

The term “alkylene” by itself or as part of another substituent means adivalent (diradical) alkyl group, wherein alkyl is defined herein.“Alkylene” is exemplified, but not limited, by —CH₂H₂H₂H₂—. Typically,an “alkylene” group will have from 1 to 24 carbon atoms, for example,having 10 or fewer carbon atoms (e.g., 1 to 8 or 1 to 6 carbon atoms). A“lower alkylene” group is an alkylene group having from 1 to 4 carbonatoms.

The term “alkenyl” by itself or as part of another substituent refers toa straight or branched chain hydrocarbon radical having from 2 to 24carbon atoms and at least one double bond. A typical alkenyl group hasfrom 2 to 10 carbon atoms and at least one double bond. In oneembodiment, alkenyl groups have from 2 to 8 carbon atoms or from 2 to 6carbon atoms and from 1 to 3 double bonds. Exemplary alkenyl groupsinclude vinyl, 2-propenyl, 1-but-3-enyl, crotyl, 2-(butadienyl),2,4-pentadienyl, 3-(1,4-pentadienyl), 2-isopentenyl, 1-pent-3-enyl,1-hex-5-enyl and the like.

The term “alkynyl” by itself or as part of another substituent refers toa straight or branched chain, unsaturated or polyunsaturated hydrocarbonradical having from 2 to 24 carbon atoms and at least one triple bond. Atypical “alkynyl” group has from 2 to 10 carbon atoms and at least onetriple bond. In one aspect of the disclosure, alkynyl groups have from 2to 6 carbon atoms and at least one triple bond. Exemplary alkynyl groupsinclude prop-1-ynyl, prop-2-ynyl (i.e., propargyl), ethynyl and3-butynyl.

The terms “alkoxy,” “alkylamino” and “alkylthio” (or thioalkoxy) areused in their conventional sense, and refer to alkyl groups that areattached to the remainder of the molecule via an oxygen atom, an aminogroup, or a sulfur atom, respectively.

The term “heteroalkyl,” by itself or in combination with another term,means a stable, straight or branched chain hydrocarbon radicalconsisting of the stated number of carbon atoms (e.g., C₂-C₁₀, or C₂-C₈)and at least one heteroatom chosen, e.g., from N, O S, Si, B and P (inone embodiment, N, O and S), wherein the nitrogen, sulfur and phosphorusatoms are optionally oxidized, and the nitrogen atom(s) are optionallyquaternized. The heteroatom(s) is/are placed at any interior position ofthe heteroalkyl group. Examples of heteroalkyl groups include, but arenot limited to, —CH₂—CH₂—O—CH₃, —CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃,—CH₂—S—CH₂—CH₃, —CH₂—CH₂—S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃,—CH₂—Si(CH₃)₃, —CH₂—CH═N—OCH₃, and —CH═CH—N(CH₃)—CH₃. Up to twoheteroatoms can be consecutive, such as, for example, —CH₂—NH—OCH₃ and—CH₂—O—Si(CH₃)₃.

Similarly, the term “heteroalkylene” by itself or as part of anothersubstituent means a divalent radical derived from heteroalkyl, asexemplified, but not limited by, —CH₂—CH₂—S—CH₂—CH₂— and—CH₂—S—CH₂—CH₂—NH—CH₂—. Typically, a heteroalkyl group will have from 3to 24 atoms (carbon and heteroatoms, excluding hydrogen) (3- to24-membered heteroalkyl). In another example, the heteroalkyl group hasa total of 3 to 10 atoms (3- to 10-membered heteroalkyl) or from 3 to 8atoms (3- to 8-membered heteroalkyl). The term “heteroalkyl” includes“heteroalkylene” wherever appropriate, e.g., when the formula indicatesthat the heteroalkyl group is divalent or when substituents are joinedto form a ring.

The term “cycloalkyl” by itself or in combination with other terms,represents a saturated or unsaturated, non-aromatic carbocyclic radicalhaving from 3 to 24 carbon atoms, for example, having from 3 to 12carbon atoms (e.g., C₃-C₈ cycloalkyl or C₃-C₆ cycloalkyl). Examples ofcycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cycloheptyl, 1-cyclohexenyl, 3-cyclohexenyl,cycloheptyl and the like. The term “cycloalkyl” also includes bridged,polycyclic (e.g., bicyclic) structures, such as norbornyl, adamantyl andbicyclo[2.2.1]heptyl. The “cycloalkyl” group can be fused to at leastone (e.g., 1 to 3) other ring selected from aryl (e.g., phenyl),heteroaryl (e.g., pyridyl) and non-aromatic (e.g., carbocyclic orheterocyclic) rings. When the “cycloalkyl” group includes a fused aryl,heteroaryl or heterocyclic ring, then the “cycloalkyl” group is attachedto the remainder of the molecule via the carbocyclic ring.

The term “heterocycloalkyl,” “heterocyclic,” “heterocycle,” or“heterocyclyl,” by itself or in combination with other terms, representsa carbocyclic, non-aromatic ring (e.g., 3- to 8-membered ring and forexample, 4-, 5-, 6- or 7-membered ring) containing at least one and upto 5 heteroatoms selected from, e.g., N, O S, Si, B and P (for example,N, O and S), wherein the nitrogen, sulfur and phosphorus atoms areoptionally oxidized, and the nitrogen atom(s) are optionally quaternized(e.g., from 1 to 4 heteroatoms selected from nitrogen, oxygen andsulfur), or a fused ring system of 4- to 8-membered rings, containing atleast one and up to 10 heteroatoms (e.g., from 1 to 5 heteroatomsselected from N, O and S) in stable combinations known to those of skillin the art. Exemplary heterocycloalkyl groups include a fused phenylring. When the “heterocyclic” group includes a fused aryl, heteroaryl orcycloalkyl ring, then the “heterocyclic” group is attached to theremainder of the molecule via a heterocycle. A heteroatom can occupy theposition at which the heterocycle is attached to the remainder of themolecule.

Exemplary heterocycloalkyl or heterocyclic groups of the presentdisclosure include morpholinyl, thiomorpholinyl, thiomorpholinylS-oxide, thiomorpholinyl S,S-dioxide, piperazinyl, homopiperazinyl,pyrrolidinyl, pyrrolinyl, imidazolidinyl, tetrahydropyranyl,piperidinyl, tetrahydrofuranyl, tetrahydrothienyl, piperidinyl,homopiperidinyl, homomorpholinyl, homothiomorpholinyl,homothiomorpholinyl S,S-dioxide, oxazolidinonyl, dihydropyrazolyl,dihydropyrrolyl, dihydropyrazolyl, dihydropyridyl, dihydropyrimidinyl,dihydrofuryl, dihydropyranyl, tetrahydrothienyl S-oxide,tetrahydrothienyl S,S-dioxide, homothiomorpholinyl S-oxide,1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,1-piperazinyl, 2-piperazinyl, and the like.

By “aryl” is meant a 5-, 6- or 7-membered, aromatic carbocyclic grouphaving a single ring (e.g., phenyl) or being fused to other aromatic ornon-aromatic rings (e.g., from 1 to 3 other rings). When the “aryl”group includes a non-aromatic ring (such as in1,2,3,4-tetrahydronaphthyl) or heteroaryl group then the “aryl” group isbonded to the remainder of the molecule via an aryl ring (e.g., a phenylring). The aryl group is optionally substituted (e.g., with 1 to 5substituents described herein). In one example, the aryl group has from6 to 10 carbon atoms. Non-limiting examples of aryl groups includephenyl, 1-naphthyl, 2-naphthyl, quinoline, indanyl, indenyl,dihydronaphthyl, fluorenyl, tetralinyl, benzo[d][1,3]dioxolyl or6,7,8,9-tetrahydro-5H-benzo[a]cycloheptenyl. In one embodiment, the arylgroup is selected from phenyl, benzo[d][1,3]dioxolyl and naphthyl. Thearyl group, in yet another embodiment, is phenyl.

The term “arylalkyl” or “aralkyl” is meant to include those radicals inwhich an aryl group or heteroaryl group is attached to an alkyl group tocreate the radicals -alkyl-aryl and -alkyl-heteroaryl, wherein alkyl,aryl and heteroaryl are defined herein. Exemplary “arylalkyl” or“aralkyl” groups include benzyl, phenethyl, pyridylmethyl and the like.

By “aryloxy” is meant the group-O-aryl, where aryl is as defined herein.In one example, the aryl portion of the aryloxy group is phenyl ornaphthyl. The aryl portion of the aryloxy group, in one embodiment, isphenyl.

The term “heteroaryl” or “heteroaromatic” refers to a polyunsaturated,5-, 6- or 7-membered aromatic moiety containing at least one heteroatom(e.g., 1 to 5 heteroatoms, such as 1-3 heteroatoms) selected from N, O,S, Si and B (for example, N, O and S), wherein the nitrogen and sulfuratoms are optionally oxidized, and the nitrogen atom(s) are optionallyquaternized. The “heteroaryl” group can be a single ring or be fused toother aryl, heteroaryl, cycloalkyl or heterocycloalkyl rings (e.g., from1 to 3 other rings). When the “heteroaryl” group includes a fused aryl,cycloalkyl or heterocycloalkyl ring, then the “heteroaryl” group isattached to the remainder of the molecule via the heteroaryl ring. Aheteroaryl group can be attached to the remainder of the moleculethrough a carbon- or heteroatom.

In one example, the heteroaryl group has from 4 to 10 carbon atoms andfrom 1 to 5 heteroatoms selected from O, S and N. Non-limiting examplesof heteroaryl groups include pyridyl, pyrimidinyl, quinolinyl,benzothienyl, indolyl, indolinyl, pyridazinyl, pyrazinyl, isoindolyl,isoquinolyl, quinazolinyl, quinoxalinyl, phthalazinyl, imidazolyl,isoxazolyl, pyrazolyl, oxazolyl, thiazolyl, indolizinyl, indazolyl,benzothiazolyl, benzimidazolyl, benzofuranyl, furanyl, thienyl,pyrrolyl, oxadiazolyl, thiadiazolyl, triazolyl, tetrazolyl,isothiazolyl, naphthyridinyl, isochromanyl, chromanyl,tetrahydroisoquinolinyl, isoindolinyl, isobenzotetrahydrofuranyl,isobenzotetrahydrothienyl, isobenzothienyl, benzoxazolyl, pyridopyridyl,benzotetrahydrofuranyl, benzotetrahydrothienyl, purinyl, benzodioxolyl,triazinyl, pteridinyl, benzothiazolyl, imidazopyridyl, imidazothiazolyl,dihydrobenzisoxazinyl, benzisoxazinyl, benzoxazinyl,dihydrobenzisothiazinyl, benzopyranyl, benzothiopyranyl, chromonyl,chromanonyl, pyridyl-N-oxide, tetrahydroquinolinyl, dihydroquinolinyl,dihydroquinolinonyl, dihydroisoquinolinonyl, dihydrocoumarinyl,dihydroisocoumarinyl, isoindolinonyl, benzodioxanyl, benzoxazolinonyl,pyrrolyl N-oxide, pyrimidinyl N-oxide, pyridazinyl N-oxide, pyrazinylN-oxide, quinolinyl N-oxide, indolyl N-oxide, indolinyl N-oxide,isoquinolyl N-oxide, quinazolinyl N-oxide, quinoxalinyl N-oxide,phthalazinyl N-oxide, imidazolyl N-oxide, isoxazolyl N-oxide, oxazolylN-oxide, thiazolyl N-oxide, indolizinyl N-oxide, indazolyl N-oxide,benzothiazolyl N-oxide, benzimidazolyl N-oxide, pyrrolyl N-oxide,oxadiazolyl N-oxide, thiadiazolyl N-oxide, triazolyl N-oxide, tetrazolylN-oxide, benzothiopyranyl S-oxide, benzothiopyranyl S,S-dioxide.Exemplary heteroaryl groups include imidazolyl, pyrazolyl, thiadiazolyl,triazolyl, isoxazolyl, isothiazolyl, imidazolyl, thiazolyl, oxadiazolyl,and pyridyl. Other exemplary heteroaryl groups include 1-pyrrolyl,2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl,pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl,3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl,5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl,3-pyridyl, pyridin-4-yl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl,purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl,2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl. Substituentsfor each of the above noted aryl and heteroaryl ring systems areselected from acceptable aryl group substituents described below.

C. Protease-Cleavable Substrate (Zy)

The procoagulant compounds of the invention comprise aprotease-cleavable substrate (Zy) linked to a self-immolative spacer(Bx). In some embodiments, the procoagulant compound of the inventioncomprises a single protease-cleavable substrate. In other embodiments,particularly embodiments where more that one procoagulant polypeptide(e.g., a clotting factor o procoagulant peptide) or one or moreheterologous moieties comprising polypeptide sequences are present,additional protease-cleavable substrate moieties (alone or in tandemwith self-immolative spacers), can be linked to the N-terminus of thesequence of the procoagulant polypeptide or heterogous moiety. In someembodiments, protease-cleavable substrate moieties (alone or in tandemwith self-immolative spacers), can be linked to the N-terminus oflinkers (L) comprising peptide linkers.

Accordingly, procoagulant compounds of the invention can comprise aZy-Bx-Pep1 module and further comprise, e.g., one or more of thefollowing additional modules:

Zy-Het (wherein Het is Het1 or Het2),

Zy-Bx-Het (wherein Het is Het1 or Het2),

Zy-Pep (wherein Pep is Pep2 or an additional polypeptide if theprocoagulant compound comprises more than two polypeptides)

Zy-Bx-Pep (wherein Pep is Pep2 or an additional polypeptide if theprocoagulant compound comprises more than two polypeptides), wherein theadditional module is covalently linked to the N-terminus or theC-terminus of the Zy-Bx-Pep1 module, with optionally one or more linkersor heterologous moieties interposed between the Zy-Bx-Pep1 and theadditional module.

In some embodiments, when more that one protease-cleavable substrate ispresent, the substrates can be cleaved by the same or by differentproteases. In embodiments where the protease-cleavable substrates arecleaved by the same protease, the protease-cleavable substrate can bethe same or they can be different.

In some embodiments, the protease-cleavable substrate is a selectivesubstrate for enzymatic cleavage by one or more proteases, e.g.,blood-coagulation cascade proteases. Blood-coagulating cascade proteasesinclude, but are not necessarily limited to, thrombin, FVIIa, FIXa, FXa,FXIa, and FXIIa.

The term “blood-coagulation cascade” used herein refers to theintrinsic, extrinsic, and common pathways. The intrinsic coagulationpathway leads to the formation of FIXa, that in conjunction with FVIIIaand FX, phospholipid and Ca2+ gives FXa. The extrinsic pathway gives FXaand FIXa after the combination of tissue factor and FVII. The commoncoagulation pathway interacts with the blood coagulation factors FV,FVIII, FIX and FX to cleave prothrombin to thrombin (FIIa), which isthen able to cleave fibrinogen to fibrin.

In some embodiments, the protease-cleavable substrate comprise acleavage site for a protease selected from neprilysin (CALLA or CDlO),thimet oligopeptidase (TOP), leukotriene A4 hydrolase, endothelinconverting enzymes, ste24 protease, neurolysin, mitochondrialintermediate peptidase, interstitial collagenases, collagenases,stromelysins, macrophage elastase, matrilysin, gelatinases, meprins,procollagen C-endopeptidases, procollagen N-endopeptidases, ADAMs andADAMTs metalloproteinases, myelin associated metalloproteinases.enamelysin, tumor necrosis factor α-converting enzyme, insulysin,nardilysin, mitochondrial processing peptidase, magnolysin,dactylysin-like metal loproteases, neutrophil collagenase, matrixmetallopeptidases, membrane-type matrix metalloproteinases, SP2endopeptidase, prostate specific antigen (PSA), plasmin, urokinase,human fibroblast activation protein (FAPα), trypsin, chymotrypsins,caldecrin, pancreatic elastases, pancreatic endopeptidase,enteropeptidase, leukocyte elastase, myeloblasts, chymases, tryptase,granzyme, stratum corneum chymotryptic enzyme, acrosin, kallikreins,complement components and factors, alternative-complement pathway c3/c5convertase, mannose-binding protein-associated serine protease,coagulation factors, thrombin, protein c, u and t-type plasminogenactivator, cathepsin G, hepsin, prostasin, hepatocyte growthfactor-activating endopeptidase, subtilisin/kexin type proproteinconvertases, furin, proprotein convertases, prolyl peptidases,acylaminoacyl peptidase, peptidyl-glycaminase, signal peptidase,n-terminal nucleophile aminohydrolases, 20s proteasome, γ-glutamyltranspeptidase, mitochondrial endopeptidase, mitochondrial endopeptidaseIa, htra2 peptidase, matriptase, site 1 protease, legumain, cathepsins,cysteine cathepsins, calpains, ubiquitin isopeptidase T, caspases,glycosylphosphatidylinositoliprotein transamidase, cancer procoagulant,prohormone thiol protease, γ-Glutamyl hydrolase, bleomycin hydrolase,seprase, cathepsin D, pepsins, chymosyn, gastricsin, renin, yapsinand/or memapsins, Prostate-Specific antigen (PSA), or any combinationsthereof. See, e.g., Kohchi et al. Bioorganic & Medicinal ChemistryLetters 17:2241-2245 (2007); Brady et al. J. Med. Chem. 45:4706-4715(2002).

In specific embodiments, the protease-cleavable substrate is selectivelycleaved by thrombin at the site of injury. In some embodiments, theprotease-cleavable substrate is selectively cleaved by thrombin invitro, for example, when the procoagulant peptides of the invention areused for diagnosis or visualization. Non-limiting exemplarythrombin-cleavable substrates include, e.g., DFLAEGGGVR (SEQ ID NO: 4),TTKIKPR (SEQ ID NO: 5), or LVPRG (SEQ ID NO: 6), or a sequencecomprising, consisting essentially of, or consisting of ALRPR (SEQ IDNO: 7) (e.g., ALRPRVVGGA (SEQ ID NO: 16)).

In specific embodiments, the thrombin-cleavable substrate comprises thesequence ALRPR (SEQ ID NO: 7), ALVPR (SEQ ID NO: 17), LVPR (SEQ ID NO:18), D-Phe-Pro-Arg (SEQ ID NO: 19), D-Ala-Leu-Val-Pro-Arg (SEQ ID NO:20), or D-Phe-Pip-Arg (Pip=pipecolic acid) (SEQ ID NO: 21) (see, e.g.,Tung et al., ChemBioChem 3:207-2011 (2002); Jaffer et al. Arterioscler.Thromb. Vasc. Biol. 22:1929-1935 (2002); Rijkers et al. ThrombosisResearch 79: 491-499 (1995)). Numerous synthetic thrombin-cleavablesubstrates are known in the art (see, e.g., Izquierdo & Burguillo, Int.J. Biochem. 21:579-592 (1989); WO1992/007869). The consensus cleavagesite for thrombin has been identified as P3-hydrophobic, P2-Pro, Pip,P3-Arg, or isosteric and isolectronic with Arg; e.g., D-Phe-Pip-Lys (SEQID NO: 22), D-Phe-Pro-Lys (SEQ ID NO: 23), D-Phe-Pip-Orn (SEQ ID NO:24). See also, Gallwitz et al. PLoS ONE 7(2): e31756.doi:10.1371/journal.pone.0031756 (2012); Tanihara et al. Peptides19:421-425 (1998); Rijkers et al. Thrombosis 79:491-499 (1995).

In some embodiments, the protease-cleavable substrate comprises a FXIacleavage site (e.g., KLTRIAET (SEQ ID NO: 25)), a FXIa cleavage site(e.g., DFTR↓VVG (SEQ ID NO: 26)), a FXIIa cleavage site (e.g., TMTR↓IVGG(SEQ ID NO: 27)), a kallikrein cleavage site (e.g., SPFRISTGG (SEQ IDNO: 28)), a FVIIa cleavage site (e.g., LQVR↓IVGG (SEQ ID NO: 29)), aFIXa cleavage site (e.g., PLGR↓IVGG (SEQ ID NO: 30)), a FXa cleavagesite (e.g., IEGR↓TVGG (SEQ ID NO: 31)), a FIIa (thrombin) cleavage site(e.g., LTPR₁SLLV (SEQ ID NO: 32)), a Elastase-2 cleavage site (e.g.,LGPV↓SGVP (SEQ ID NO: 33)), a Granzyme-B cleavage site (e.g, VAGD↓SLEE(SEQ ID NO: 34)), a MMP-12 cleavage site (e.g., GPAG↓LGGA (SEQ ID NO:35)), a MMP-13 cleavage site (e.g., GPAG↓LRGA (SEQ ID NO: 36)), a MMP-17cleavage site (e.g., APLG↓LRLR (SEQ ID NO: 37)), a MMP-20 cleavage site(e.g., PALP↓LVAQ (SEQ ID NO: 38)), a TEV cleavage site (e.g., ENLYFQ↓G(SEQ ID NO: 39)), a Enterokinase cleavage site (e.g., DDDK↓IVGG (SEQ IDNO: 40)), a Protease 3 (PRESCISSION™) cleavage site (e.g., LEVLFQ↓GP(SEQ ID NO: 41)), and a Sortase A cleavage site (e.g., LPKT↓GSES) (SEQID NO: 42). In certain embodiments, the FXIa cleavage sites include, butare not limited to, e.g., TQSFNDFTR (SEQ ID NO: 2) and SVSQTSKLTR (SEQID NO: 3).

D. Linkers (L)

As described above, the procoagulant compounds of the invention cancomprise one or more linkers. As used herein, the term “linker”(represented as L in the formulas disclosed herein) refers to a peptideor polypeptide sequence (e.g., a synthetic peptide or polypeptidesequence), or a non-peptide linker for which its main function is toconnect two moieties (e.g., Het1, Het2, Pep1, Pep2, Bx) in aprocoagulant compound of the invention. A linker can be present betweenany two moieties or non-linker elements of the procoagulant compounds ofthe invention. For example, one or more linkers can be present between aprotease-cleavable substrate (e.g., a thrombin-cleavable substrate) anda heterologous moiety, or between a protease-cleavable substrate and apolypeptide (e.g., a procoagulant peptide, a clotting factor, or anon-procoagulant polypeptide), or between a first polypeptide and asecond polypeptide, or between a first heterologous moiety and a secondheterologous moiety. In some embodiments, two or more linkers can belinked in tandem.

When multiple linkers are present in a procoagulant compound of theinvention, each of the linkers can be the same or different. Generally,linkers provide flexibility to the polypeptide molecule. Linkers are nottypically cleaved; however in certain embodiments, such cleavage can bedesirable. Accordingly, in some embodiments a linker can comprise one ormore protease-cleavable sites, which can be located within the sequenceof the linker or flanking the linker at either end of the linkersequence.

In one embodiment, the linker is a peptide linker. In some embodiments,the peptide linker can comprise at least two amino, at least three, atleast four, at least five, at least 10, at least 20, at least 30, atleast 40, at least 50, at least 60, at least 70, at least 80, at least90, or at least 100 amino acids. In other embodiments, the peptidelinker can comprise at least 200, at least 300, at least 400, at least500, at least 600, at least 700, at least 800, at least 900, or at least1,000 amino acids. In some embodiments, the peptide linker can compriseat least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300,400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600,1700, 1800, 1900, or 2000 amino acids. The peptide linker can comprise1-5 amino acids, 1-10 amino acids, 1-20 amino acids, 10-50 amino acids,50-100 amino acids, 100-200 amino acids, 200-300 amino acids, 300-400amino acids, 400-500 amino acids, 500-600 amino acids, 600-700 aminoacids, 700-800 amino acids, 800-900 amino acids, or 900-1000 aminoacids.

Examples of peptide linkers are well known in the art, for examplepeptide linkers according to the formula [(Gly)_(x)-Ser_(y)]_(z) where xis from 1 to 4, y is 0 or 1, and z is from 1 to 50. In one embodiment,the peptide linker comprises the sequence G_(n), where n can be aninteger from 1 to 100. In a specific embodiment, the specificembodiment, the sequence of the peptide linker is GGGG. The peptidelinker can comprise the sequence (GA)_(n). The peptide linker cancomprise the sequence (GGS)_(n). In other embodiments, the peptidelinker comprises the sequence (GGGS)_(n) (SEQ ID NO: 43). In still otherembodiments, the peptide linker comprises the sequence(GGS)_(n)(GGGGS)_(n) (SEQ ID NO: 44). In these instances, n can be aninteger from 1-100. In other instances, n can be an integer from 1-20,i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,or 20. Examples of linkers include, but are not limited to, GGG, SGGSGGS(SEQ ID NO: 45), GGSGGSGGSGGSGGG (SEQ ID NO:46), GGSGGSGGGGSGGGGS (SEQID NO:47), GGSGGSGGSGGSGGSGGS (SEQ ID NO:48), or GGGGSGGGGSGGGGS (SEQ IDNO:49). In other embodiments, the linker is a poly-G sequence (GGGG)_(n)(SEQ ID NO: 50), where n can be an integer from 1-100.

In one embodiment, the peptide linker is synthetic, i.e., non-naturallyoccurring. In one embodiment, a peptide linker includes peptides (orpolypeptides) (e.g., natural or non-naturally occurring peptides) whichcomprise an amino acid sequence that links or genetically fuses a firstlinear sequence of amino acids to a second linear sequence of aminoacids to which it is not naturally linked or genetically fused innature. For example, in one embodiment the peptide linker can comprisenon-naturally occurring polypeptides which are modified forms ofnaturally occurring polypeptides (e.g., comprising a mutation such as anaddition, substitution or deletion). In another embodiment, the peptidelinker can comprise non-naturally occurring amino acids. In anotherembodiment, the peptide linker can comprise naturally occurring aminoacids occurring in a linear sequence that does not occur in nature. Instill another embodiment, the peptide linker can comprise a naturallyoccurring polypeptide sequence.

In some embodiments, the linker comprises a non-peptide linker. In otherembodiments, the linker consists of a non-peptide linker. In someembodiments, the non-peptide linker can be, e.g., maleimido caproyl(MC), maleimido propanoyl (MP), methoxyl polyethyleneglycol (MPEG),succinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC),m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), succinimidyl4-(p-maleimidophenyl)butyrate (SMPB),N-succinimidyl(4-iodoacetyl)aminobenzonate (SIAB), succinimidyl6-[3-(2-pyridyldithio)-propionamide]hexanoate (LC-SPDP),4-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pyridyldithio)toluene(SMPT), etc. (see, e.g., U.S. Pat. No. 7,375,078).

Linkers can be introduced into polypeptide sequences using techniquesknown in the art (e.g., chemical conjugation, recombinant techniques, orpeptide synthesis). Modifications can be confirmed by DNA sequenceanalysis. In some embodiments, the linkers can be introduced usingrecombinant techniques. In other embodiments, the linkers can beintroduced using solid phase peptide synthesis. In certain embodiments,a procoagulant compound of the invention can contain simultaneously oneor more linkers that have been introduced using recombinant techniquesand one or more linkers that have been introduced using solid phasepeptide synthesis or methods of chemical conjugation known in the art.

E. Heterologous Moieties (e.g., Het1, Het2, . . . , Het_(n))

In some embodiments, the procoagulant compound of the invention cancomprise one heterologous moiety (indicated herein as “Het1” or “Het2”).In other embodiments, the procoagulant compound of the invention cancomprise two heterologous moieties (“Het1” and “Het2”). In yet otherembodiments, the procoagulant compound of the invention can comprisemore than two heterologous moieties, e.g., three, four, five, or morethan five heterologous moieties. In some embodiments, all theheterologous moieties are identical. In some embodiments, at least oneheterologous moiety is different from the other heterologous moieties.In some embodiments, the procoagulant compound of the invention cancomprise two, three or more than three heterologous moieties in tandem.In other embodiments, the procoagulant compound of the invention cancomprise two, three, or more than heterologous moieties wherein at leastan additional moiety (e.g., a procoagulant polypeptide, a linker, aprotease-cleavable substrate, a self-immolative spacer, or combinationsthereof) is interposed between two heterologous moieties.

A heterologous moiety can comprise a heterologous polypeptide moiety, ora heterologous non-polypeptide moiety, or both. In one specificembodiment, Het1 is a first heterologous moiety, e.g., a half-lifeextending molecule which is known in the art. In some embodiments, Het2is a second heterologous moiety that can also be a half-life extendingmolecule which is known in the art. In some aspects, the heterologousmoiety comprises a combination of a heterologous polypeptide and anon-polypeptide moiety.

In certain embodiments, the first heterologous moiety (e.g., a first Fcregion) and the second heterologous moiety (e.g., a second Fc region)are associated with each other to form a dimer. In one embodiment, thesecond heterologous moiety is a second Fc region, wherein the second Fcregion is linked to or associated with the first heterologous moiety,e.g., the first Fc region. For example, the second heterologous moiety(e.g., the second Fc region) can be linked to the first heterologousmoiety (e.g., the first Fc region) by a linker or associated with thefirst heterologous moiety by a covalent or non-covalent bond.

In some embodiments, the Het1 and Het2 heterologous moieties arepeptides and polypeptides with either unstructured or structuredcharacteristics that are associated with the prolongation of in vivohalf-life when incorporated in a procoagulant compound of the invention.Non-limiting examples include albumin, albumin fragments, Fc fragmentsof immunoglobulins, the β subunit of the C-terminal peptide (CTP) of theβ subunit of human chorionic gonadotropin, a HAP sequence, XTEN, atransferrin or a fragment thereof, a PAS polypeptide, polyglycinelinkers, polyserine linkers, albumin-binding moieties, or any fragments,derivatives, variants, or combinations of these polypeptides. In otherrelated aspects a heterologous moiety can include an attachment site(e.g., a cysteine amino acid) for a non-polypeptide moiety such aspolyethylene glycol (PEG), hydroxyethyl starch (HES), polysialic acid,or any derivatives, variants, or combinations of these elements. In someaspects, a heterologous moiety consisting of a cysteine amino acid thatfunction as an attachment site for a non-polypeptide moiety such aspolyethylene glycol (PEG), hydroxyethyl starch (HES), polysialic acid,XTEN, or any derivatives, variants, or combinations of these elements.

In some embodiments, the heterologous moiety is a polypeptidecomprising, consisting essentially of, or consisting of at least about10, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300,1400, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, or 4000 aminoacids. In other embodiments, the heterologous moiety is a polypeptidecomprising, consisting essentially of, or consisting of about 100 toabout 200 amino acids, about 200 to about 300 amino acids, about 300 toabout 400 amino acids, about 400 to about 500 amino acids, about 500 toabout 600 amino acids, about 600 to about 700 amino acids, about 700 toabout 800 amino acids, about 800 to about 900 amino acids, or about 900to about 1000 amino acids.

In certain embodiments, a heterologous moiety improves one or morepharmacokinetic properties of the procoagulation compound withoutsignificantly affecting the biological activity or function of the Pep1and/or Pep2 polypeptides (e.g., procoagulant activity of a clottingfactor or a fragment thereof, or of procoagulant activity of aprocoagulant synthetic peptide).

In certain embodiments, a heterologous moiety increases the in vivoand/or in vitro half-life of the procoagulant compound of the invention.In other embodiments, a heterologous moiety facilitates visualization orlocalization of the procoagulant compound of the invention or a fragmentthereof (e.g., a fragment comprising a heterologous moiety afterproteolytic cleavage of the protease-cleavable substrate Zy).Visualization and/or location of the procoagulant compound of theinvention or a fragment thereof can be in vivo, in vitro, ex vivo, orcombinations thereof.

In other embodiments, a heterologous moiety increases stability of theprocoagulant compound of the invention or a fragment thereof (e.g., afragment comprising a heterologous moiety after proteolytic cleavage ofthe protease-cleavable substrate Zy). As used herein, the term“stability” refers to an art-recognized measure of the maintenance ofone or more physical properties of the procoagulant compound in responseto an environmental condition (e.g., an elevated or loweredtemperature). In certain aspects, the physical property can be themaintenance of the covalent structure of the procoagulant compound(e.g., the absence of proteolytic cleavage, unwanted oxidation ordeamidation). In other aspects, the physical property can also be thepresence of the procoagulant compound in a properly folded state (e.g.,the absence of soluble or insoluble aggregates or precipitates). In oneaspect, the stability of the procoagulant compound is measured byassaying a biophysical property of the procoagulant compound, forexample thermal stability, pH unfolding profile, stable removal ofglycosylation, solubility, biochemical function (e.g., ability to bindto a protein, receptor or ligand), etc., and/or combinations thereof. Inanother aspect, biochemical function is demonstrated by the bindingaffinity of the interaction. In one aspect, a measure of proteinstability is thermal stability, i.e., resistance to thermal challenge.Stability can be measured using methods known in the art, such as, HPLC(high performance liquid chromatography), SEC (size exclusionchromatography), DLS (dynamic light scattering), etc. Methods to measurethermal stability include, but are not limited to differential scanningcalorimetry (DSC), differential scanning fluorimetry (DSF), circulardichroism (CD), and thermal challenge assay.

1. Half-Life Extending Heterologous Moieties

In certain aspects, a procoagulant compound of the invention comprisesat least one half-life extending moiety, i.e., a heterologous moietywhich increases the in vivo half-life of the procoagulant compound withrespect to the in vivo half-life of the corresponding procoagulantcompound lacking such heterologous moiety. In vivo half-life of aprocoagulant compound can be determined by any method known to those ofskill in the art, e.g., activity assays (chromogenic assay or one stageclotting aPTT assay), ELISA, etc.

In some embodiments, the presence of one or more half-life extendingmoieties results in the half-life of the procoagulant compound to beincreased compared to the half life of the corresponding procoagulantcompound lacking such one or more half-life extending moieties. Thehalf-life of the procoagulant compound comprising a half-life extendingmoiety is at least about 1.5 times, at least about 2 times, at leastabout 2.5 times, at least about 3 times, at least about 4 times, atleast about 5 times, at least about 6 times, at least about 7 times, atleast about 8 times, at least about 9 times, at least about 10 times, atleast about 11 times, or at least about 12 times longer than the in vivohalf-life of the corresponding procoagulant compound lacking suchhalf-life extending moiety.

In one embodiment, the half-life of the procoagulant compound comprisinga half-life extending moiety is about 1.5-fold to about 20-fold, about1.5 fold to about 15 fold, or about 1.5 fold to about 10 fold longerthan the in vivo half-life of the corresponding procoagulant compoundlacking such half-life extending moiety. In another embodiment, thehalf-life of procoagulant compound comprising a half-life extendingmoiety is extended about 2-fold to about 10-fold, about 2-fold to about9-fold, about 2-fold to about 8-fold, about 2-fold to about 7-fold,about 2-fold to about 6-fold, about 2-fold to about 5-fold, about 2-foldto about 4-fold, about 2-fold to about 3-fold, about 2.5-fold to about10-fold, about 2.5-fold to about 9-fold, about 2.5-fold to about 8-fold,about 2.5-fold to about 7-fold, about 2.5-fold to about 6-fold, about2.5-fold to about 5-fold, about 2.5-fold to about 4-fold, about 2.5-foldto about 3-fold, about 3-fold to about 10-fold, about 3-fold to about9-fold, about 3-fold to about 8-fold, about 3-fold to about 7-fold,about 3-fold to about 6-fold, about 3-fold to about 5-fold, about 3-foldto about 4-fold, about 4-fold to about 6 fold, about 5-fold to about7-fold, or about 6-fold to about 8 fold as compared to the in vivohalf-life of the corresponding procoagulant compound lacking suchhalf-life extending moiety.

In other embodiments, the half-life of the procoagulant compoundcomprising a half-life extending moiety is at least about 17 hours, atleast about 18 hours, at least about 19 hours, at least about 20 hours,at least about 21 hours, at least about 22 hours, at least about 23hours, at least about 24 hours, at least about 25 hours, at least about26 hours, at least about 27 hours, at least about 28 hours, at leastabout 29 hours, at least about 30 hours, at least about 31 hours, atleast about 32 hours, at least about 33 hours, at least about 34 hours,at least about 35 hours, at least about 36 hours, at least about 48hours, at least about 60 hours, at least about 72 hours, at least about84 hours, at least about 96 hours, or at least about 108 hours.

In still other embodiments, the half-life of the procoagulant compoundcomprising a half-life extending moiety is about 15 hours to about twoweeks, about 16 hours to about one week, about 17 hours to about oneweek, about 18 hours to about one week, about 19 hours to about oneweek, about 20 hours to about one week, about 21 hours to about oneweek, about 22 hours to about one week, about 23 hours to about oneweek, about 24 hours to about one week, about 36 hours to about oneweek, about 48 hours to about one week, about 60 hours to about oneweek, about 24 hours to about six days, about 24 hours to about fivedays, about 24 hours to about four days, about 24 hours to about threedays, or about 24 hours to about two days.

In some embodiments, the average half-life per subject of theprocoagulant compound comprising a half-life extending moiety is about15 hours, about 16 hours, about 17 hours, about 18 hours, about 19hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours,about 24 hours (1 day), about 25 hours, about 26 hours, about 27 hours,about 28 hours, about 29 hours, about 30 hours, about 31 hours, about 32hours, about 33 hours, about 34 hours, about 35 hours, about 36 hours,about 40 hours, about 44 hours, about 48 hours (2 days), about 54 hours,about 60 hours, about 72 hours (3 days), about 84 hours, about 96 hours(4 days), about 108 hours, about 120 hours (5 days), about six days,about seven days (one week), about eight days, about nine days, about 10days, about 11 days, about 12 days, about 13 days, or about 14 days.

(a) Low Complexity Polypeptides

In certain aspects, a procoagulant compound of the invention comprisesat least one heterologous moiety comprising a polypeptide with lowcompositional and/or structural complexity (e.g., a disorderedpolypeptide with no secondary or tertiary structure in solution underphysiologic conditions).

(b) CTP

In certain aspects, a procoagulant compound of the invention comprisesat least a heterologous moiety comprising one C-terminal peptide (CTP)of the β subunit of human chorionic gonadotropin, or fragment, variant,or derivative thereof. One or more CTP peptides inserted into arecombinant protein is known to increase the in vivo half-life of thatprotein. See, e.g., U.S. Pat. No. 5,712,122, incorporated by referenceherein in its entirety.

Exemplary CTP peptides include DPRFQDSSSSKAPPPSLPSPSRLPGPSDTPIL (SEQ IDNO: 51) or SSSSKAPPPSLPSPSRLPGPSDTPILPQ. (SEQ ID NO: 52). See, e.g.,U.S. Patent Application Publication No. US 2009/0087411 A1, incorporatedby reference.

(c) Immunoglobulin Constant Region (Fc) or Portion Thereof

In certain aspects, a procoagulant compound of the invention comprisesat least one Fc region. The term “Fc” or “Fc region” as used herein,means a functional neonatal Fc receptor (FcRn) binding partnercomprising an Fc domain, variant, or fragment thereof which maintain thedesirable properties of an Fc region in a chimeric protein, e.g., anincrease in in vivo half-life. Myriad mutants, fragments, variants, andderivatives are described, e.g., in PCT Publication Nos. WO 2011/069164A2, WO 2012/006623 A2, WO 2012/006635 A2, or WO 2012/006633 A2, all ofwhich are incorporated herein by reference in their entireties. An Fcregion is comprised of domains denoted CH (constant heavy) domains (CH1,H2, etc.). Depending on the isotype, (i.e. IgG, IgM, IgA IgD, or IgE),the Fc region can be comprised of three or four CH domains. Someisotypes (e.g. IgG) Fc regions also contain a hinge region. See Janewayet al. 2001, Immunobiology, Garland Publishing, N.Y., N.Y.

An Fc region or a portion thereof for producing the procoagulantcompound of the present invention can be obtained from a number ofdifferent sources. In some embodiments, an Fc region or a portionthereof is derived from a human immunoglobulin. It is understood,however, that the Fc region or a portion thereof can be derived from animmunoglobulin of another mammalian species, including for example, arodent (e.g. a mouse, rat, rabbit, guinea pig) or non-human primate(e.g. chimpanzee, macaque) species. Moreover, the Fc region or a portionthereof can be derived from any immunoglobulin class, including IgM,IgG, IgD, IgA and IgE, and any immunoglobulin isotype, including IgG1,IgG2, IgG3 and IgG4. In one embodiment, the human isotype IgG1 is used.

Procoagulant compounds comprising an Fc region of an immunoglobulinbestow several desirable properties on a chimeric protein includingincreased stability, increased serum half-life (see Capon et al., 1989,Nature 337:525) as well as binding to Fc receptors such as the neonatalFc receptor (FcRn) (U.S. Pat. Nos. 6,086,875, 6,485,726, 6,030,613; WO03/077834; US2003-0235536A1), which are incorporated herein by referencein their entireties.

In certain embodiments, a procoagulant compound of the inventioncomprises one or more truncated Fc regions that are nonethelesssufficient to confer Fc receptor (FcR) binding properties to the Fcregion. For example, the portion of an Fc region that binds to FcRn(i.e., the FcRn binding portion) comprises from about amino acids282-438 of IgG1, EU numbering (with the primary contact sites beingamino acids 248, 250-257, 272, 285, 288, 290-291, 308-311, and 314 ofthe CH2 domain and amino acid residues 385-387, 428, and 433-436 of theCH3 domain. Thus, an Fc region in a procoagulant compound of theinvention can comprise or consist of an FcRn binding portion. FcRnbinding portions can be derived from heavy chains of any isotype,including IgG1, IgG2, IgG3 and IgG4. In one embodiment, an FcRn bindingportion from an antibody of the human isotype IgG1 is used. In anotherembodiment, an FcRn binding portion from an antibody of the humanisotype IgG4 is used.

In certain embodiments, an Fc region comprises at least one of: a hinge(e.g., upper, middle, and/or lower hinge region) domain (about aminoacids 216-230 of an antibody Fc region according to EU numbering), a CH2domain (about amino acids 231-340 of an antibody Fc region according toEU numbering), a CH3 domain (about amino acids 341-438 of an antibody Fcregion according to EU numbering), a CH4 domain, or a variant, portion,or fragment thereof. In other embodiments, an Fc region comprises acomplete Fc domain (i.e., a hinge domain, a CH2 domain, and a CH3domain). In some embodiments, an Fc region comprises, consistsessentially of, or consists of a hinge domain (or a portion thereof)fused to a CH3 domain (or a portion thereof), a hinge domain (or aportion thereof) fused to a CH2 domain (or a portion thereof), a CH2domain (or a portion thereof) fused to a CH3 domain (or a portionthereof), a CH2 domain (or a portion thereof) fused to both a hingedomain (or a portion thereof) and a CH3 domain (or a portion thereof).In still other embodiments, an Fc region lacks at least a portion of aCH2 domain (e.g., all or part of a CH2 domain). In a particularembodiment, an Fc region comprises or consists of amino acidscorresponding to EU numbers 221 to 447.

An Fc in a procoagulant compound of the invention can include, forexample, a change (e.g., a substitution) at one or more of the aminoacid positions disclosed in Int'l. PCT Publs. WO88/07089A1,WO96/14339A1, WO98/05787A1, WO98/23289A1, WO99/51642A1, WO99/58572A1,WO00/09560A2, WO00/32767A1, WO00/42072A2, WO02/44215A2, WO02/060919A2,WO03/074569A2, WO04/016750A2, WO04/029207A2, WO04/035752A2,WO04/063351A2, WO04/074455A2, WO04/099249A2, WO05/040217A2, WO04/044859,WO05/070963A1, WO05/077981A2, WO05/092925A2, WO05/123780A2,WO06/019447A1, WO06/047350A2, and WO06/085967A2; U.S. Pat. Publ. Nos. US2007/0231329, US2007/0231329, US2007/0237765, US2007/0237766,US2007/0237767, US2007/0243188, US2007/0248603, US2007/0286859,US2008/0057056; or U.S. Pat. Nos. 5,648,260; 5,739,277; 5,834,250;5,869,046; 6,096,871; 6,121,022; 6,194,551; 6,242,195; 6,277,375;6,528,624; 6,538,124; 6,737,056; 6,821,505; 6,998,253; 7,083,784;7,404,956, and 7,317,091, each of which is incorporated by referenceherein in its entirety. In one embodiment, the specific change (e.g.,the specific substitution of one or more amino acids disclosed in theart) can be made at one or more of the disclosed amino acid positions.In another embodiment, a different change at one or more of thedisclosed amino acid positions (e.g., the different substitution of oneor more amino acid position disclosed in the art) can be made.

An Fc region used in the invention can also comprise an art recognizedamino acid substitution which alters the glycosylation of the chimericprotein. For example, the Fc region of the procoagulant compound cancomprise an Fc region having a mutation leading to reduced glycosylation(e.g., N- or O-linked glycosylation) or can comprise an alteredglycoform of the wild-type Fc moiety (e.g., a low fucose or fucose-freeglycan).

(d) Albumin or Fragment, or Variant Thereof

In certain embodiments, the procoagulant compound of the inventioncomprises a heterologous moiety comprising albumin or a functionalfragment thereof. Human serum albumin (HSA, or HA), a protein of 609amino acids in its full-length form, is responsible for a significantproportion of the osmotic pressure of serum and also functions as acarrier of endogenous and exogenous ligands. The term “albumin” as usedherein includes full-length albumin or a functional fragment, variant,derivative, or analog thereof. Examples of albumin or the fragments orvariants thereof are disclosed in US Pat. Publ. Nos. 2008/0194481A1,2008/0004206 A1, 2008/0161243 A1, 2008/0261877 A1, or 2008/0153751 A1 orPCT Appl. Publ. Nos. 2008/033413 A2, 2009/058322 A1, or 2007/021494 A2,which are incorporated herein by reference in their entireties.

In one embodiment, the procoagulant compound of the invention comprisesalbumin, a fragment, or a variant thereof which is further linked to aheterologous moiety selected from an immunoglobulin constant region orportion thereof (e.g., an Fc region), a PAS sequence, HES, XTEN, PEG, orany combinations thereof.

(e) Albumin Binding Moiety

In certain embodiments, the heterologous moiety is an albumin bindingmoiety, which comprises an albumin binding peptide, a bacterial albuminbinding domain, an albumin-binding antibody fragment, a fatty acid, orany combinations thereof.

For example, the albumin binding protein can be a bacterial albuminbinding protein, an antibody or an antibody fragment including domainantibodies (see U.S. Pat. No. 6,696,245). An albumin binding protein,for example, can be a bacterial albumin binding domain, such as the oneof streptococcal protein G (Konig, T. and Skerra, A. (1998) J. Immunol.Methods 218, 73-83). Other examples of albumin binding peptides that canbe used as conjugation partner are, for instance, those having aCys-Xaa₁-Xaa₂-Xaa₃-Xaa₄-Cys consensus sequence, wherein Xaa₁ is Asp,Asn, Ser, Thr, or Trp; Xaa₂ is Asn, Gln, H is, Ile, Leu, or Lys; Xaa₃ isAla, Asp, Phe, Trp, or Tyr; and Xaa₄ is Asp, Gly, Leu, Phe, Ser, or Thras described in US patent application 2003/0069395 or Dennis et al.(Dennis et al. (2002) J. Biol. Chem. 277, 35035-35043).

Domain 3 from streptococcal protein G, as disclosed by Kraulis et al.,FEBS Lett. 378:190-194 (1996) and Linhult et al., Protein Sci.11:206-213 (2002) is an example of a bacterial albumin-binding domain.Examples of albumin-binding peptides include a series of peptides havingthe core sequence DICLPRWGCLW (SEQ ID NO: 53). See, e.g., Dennis et al.,J. Biol. Chem. 2002, 277: 35035-35043 (2002). Some examples ofalbumin-binding peptides are:

(SEQ ID NO: 54) RLIEDICLPRWGCLWEDD, (SEQ ID NO: 55)QRLMEDICLPRWGCLWEDDF, (SEQ ID NO: 56) QGLIGDICLPRWGCLWGDSVK, or(SEQ ID NO: 57) GEWWEDICLPRWGCLWEEED.

Examples of albumin-binding antibody fragments are disclosed in Mullerand Kontermann, Curr. Opin. Mol. Ther. 9:319-326 (2007); Roovers et al.,Cancer Immunol. Immunother. 56:303-317 (2007), and Holt et al., Prot.Eng. Design Sci., 21:283-288 (2008), which are incorporated herein byreference in their entireties. An example of such albumin binding moietyis 2-(3-maleimidopropanamido)-6-(4-(4-iodophenyl)butanamido) hexanoate(“Albu” tag) as disclosed by Trussel et al., Bioconjugate Chem.20:2286-2292 (2009).

Fatty acids, in particular long chain fatty acids (LCFA) and long chainfatty acid-like albumin-binding compounds can be used to extend the invivo half-life of procoagulant compounds of the invention. An example ofa LCFA-like albumin-binding compound is16-(1-(3-(9-(((2,5-dioxopyrrolidin-1-yloxy)carbonyloxy)-methyl)-7-sulfo-9H-fluoren-2-ylamino)-3-oxopropyl)-2,5-dioxopyrrolidin-3-ylthio)hexadecanoic acid (see, e.g., WO 2010/140148).

(f) PAS Sequence

In other embodiments, at least one heterologous moiety is a PASsequence. A PAS sequence, as used herein, means an amino acid sequencecomprising mainly alanine and serine residues or comprising mainlyalanine, serine, and proline residues, the amino acid sequence formingrandom coil conformation under physiological conditions. Accordingly,the PAS sequence is a building block, an amino acid polymer, or asequence cassette comprising, consisting essentially of, or consistingof alanine, serine, and proline which can be used as a part of theheterologous moiety in the procoagulant compound. Yet, the skilledperson is aware that an amino acid polymer also can form random coilconformation when residues other than alanine, serine, and proline areadded as a minor constituent in the PAS sequence.

The term “minor constituent” as used herein means that amino acids otherthan alanine, serine, and proline can be added in the PAS sequence to acertain degree, e.g., up to about 12%, i.e., about 12 of 100 amino acidsof the PAS sequence, up to about 10%, i.e. about 10 of 100 amino acidsof the PAS sequence, up to about 9%, i.e., about 9 of 100 amino acids,up to about 8%, i.e., about 8 of 100 amino acids, about 6%, i.e., about6 of 100 amino acids, about 5%, i.e., about 5 of 100 amino acids, about4%, i.e., about 4 of 100 amino acids, about 3%, i.e., about 3 of 100amino acids, about 2%, i.e., about 2 of 100 amino acids, about 1%, i.e.,about 1 of 100 of the amino acids. The amino acids different fromalanine, serine and proline can be selected from Arg, Asn, Asp, Cys,Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Thr, Trp, Tyr, or Val.

Under physiological conditions, the PAS sequence stretch forms a randomcoil conformation and thereby can mediate an increased in vivo and/or invitro stability to procoagulant compound. Since the random coil domaindoes not adopt a stable structure or function by itself, the biologicalactivity mediated by the Pep1 and/or Pep2 polypeptides in theprocoagulant compound is essentially preserved. In other embodiments,the PAS sequences that form random coil domain are biologically inert,especially with respect to proteolysis in blood plasma, immunogenicity,isoelectric point/electrostatic behavior, binding to cell surfacereceptors or internalization, but are still biodegradable, whichprovides clear advantages over synthetic polymers such as PEG.

Non-limiting examples of the PAS sequences forming random coilconformation comprise an amino acid sequence selected fromASPAAPAPASPAAPAPSAPA (SEQ ID NO:58), AAPASPAPAAPSAPAPAAPS (SEQ IDNO:59), APSSPSPSAPSSPSPASPSS (SEQ ID NO:60), APSSPSPSAPSSPSPASPS (SEQ IDNO: 61), SSPSAPSPSSPASPSPSSPA (SEQ ID NO: 62), AASPAAPSAPPAAASPAAPSAPPA(SEQ ID NO: 63), ASAAAPAAASAAASAPSAAA (SEQ ID NO: 64) or anycombinations thereof. Additional examples of PAS sequences are knownfrom, e.g., US Pat. Publ. No. 2010/0292130 A1 and PCT Appl. Publ. No. WO2008/155134 A1.

(g) HAP Sequence

In certain embodiments, at least one heterologous moiety is aglycine-rich homo-amino-acid polymer (HAP). The HAP sequence cancomprise a repetitive sequence of glycine, which has at least 50 aminoacids, at least 100 amino acids, 120 amino acids, 140 amino acids, 160amino acids, 180 amino acids, 200 amino acids, 250 amino acids, 300amino acids, 350 amino acids, 400 amino acids, 450 amino acids, or 500amino acids in length. In one embodiment, the HAP sequence is capable ofextending half-life of a moiety fused to or linked to the HAP sequence.Non-limiting examples of the HAP sequence includes, but are not limitedto (Gly)_(n), (Gly₄Ser)_(n) or S(Gly₄Ser)_(n), wherein n is 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In oneembodiment, n is 20, 21, 22, 23, 24, 25, 26, 26, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, or 40. In another embodiment, n is 50, 60, 70,80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200.

(h) XTEN

In certain aspects, a procoagulant compound of the invention comprisesat least one heterologous moiety comprising an XTEN polypeptide orfragment, variant, or derivative thereof. As used here “XTEN sequence”refers to extended length polypeptides with non-naturally occurring,substantially non-repetitive sequences that are composed mainly of smallhydrophilic amino acids, with the sequence having a low degree or nosecondary or tertiary structure under physiologic conditions. As aheterologous moiety, XTENs can serve as a half life extension moiety. Inaddition, XTEN can provide desirable properties including but are notlimited to enhanced pharmacokinetic parameters and solubilitycharacteristics.

The incorporation of a heterologous moiety comprising an XTEN sequenceinto a procoagulant compound of the invention can confer to theprocoagulant compound one or more of the following advantageousproperties: conformational flexibility, enhanced aqueous solubility,high degree of protease resistance, low immunogenicity, low binding tomammalian receptors, or increased hydrodynamic (or Stokes) radii.

In certain aspects, an XTEN sequence can increase pharmacokineticproperties such as longer in vivo half-life or increased area under thecurve (AUC), so that a procoagulant compound of the invention stays invivo and has procoagulant activity for an increased period of timecompared to a procoagulant compound with the same but without the XTENheterogous moiety.

Examples of XTEN sequences that can be used as heterologous moieties inprocoagulant compounds of the invention are disclosed, e.g., in U.S. PatNos. 7,855,279 and 7,846,445, U.S. Patent Publication Nos. 2009/0092582A1, 2010/0239554 A1, 2010/0323956 A1, 2011/0046060 A1, 2011/0046061 A1,2011/0077199 A1, or 2011/0172146 A1, 2013/0017997 A1, or 2012/0263701A1, or International Patent Publication Nos. WO 2010091122 A1, WO2010144502 A2, WO 2010144508 A1, WO 2011028228 A1, WO 2011028229 A1, orWO 2011028344 A2, or International Application No. PCT/US2011/48517,filed Aug. 19, 2011, each of which is incorporated by reference hereinin its entirety.

(i) Transferrin or Fragment Thereof

In certain embodiments, at least one heterologous moiety is transferrinor a fragment thereof. Any transferrin can be used to make theprocoagulant compounds of the invention. As an example, wild-type humanTf (Tf) is a 679 amino acid protein, of approximately 75 KDa (notaccounting for glycosylation), with two main domains, N (about 330 aminoacids) and C (about 340 amino acids), which appear to originate from agene duplication. See GenBank accession numbers NM001063, XM002793,M12530, XM039845, XM 039847 and 595936 (ncbi.nlm.nih.gov/), all of whichare herein incorporated by reference in their entirety. Transferrincomprises two domains, N domain and C domain. N domain comprises twosubdomains, N1 domain and N2 domain, and C domain comprises twosubdomains, C1 domain and C2 domain.

In one embodiment, the transferrin heterologous moiety includes atransferrin splice variant. In one example, a transferrin splice variantcan be a splice variant of human transferrin, e.g., Genbank AccessionAAA61140. In another embodiment, the transferrin portion of the chimericprotein includes one or more domains of the transferrin sequence, e.g.,N domain, C domain, N1 domain, N2 domain, C1 domain, C₂ domain or anycombinations thereof.

(j) Polymer, e.g., Polyethylene Glycol (PEG)

In other embodiments, at least one heterologous moiety is a solublepolymer known in the art, including, but not limited to, polyethyleneglycol, ethylene glycol/propylene glycol copolymers,carboxymethylcellulose, dextran, or polyvinyl alcohol. In someembodiments, the procoagulant compound comprising a PEG heterologousmoiety further comprises a heterologous moiety selected from animmunoglobulin constant region or portion thereof (e.g., an Fc region),a PAS sequence, HES, and albumin, fragment, or variant thereof, an XTEN,or any combinations thereof. In still other embodiments, theprocoagulant compound comprises a clotting factor or fragment thereofand a PEG heterologous moiety, wherein the procoagulant compound furthercomprises a heterologous moiety selected from an immunoglobulin constantregion or portion thereof (e.g., an Fc region), a PAS sequence, HES, andalbumin, fragment, or variant thereof, an XTEN, or any combinationsthereof.

In yet other embodiments, the procoagulant compound comprises a clottingfactor or fragment thereof, a second clotting factor or fragmentthereof, and a PEG heterologous moiety, wherein the procoagulantcompound further comprises a heterologous moiety selected from animmunoglobulin constant region or portion thereof (e.g., an Fc region),a PAS sequence, HES, and albumin, fragment, or variant thereof, an XTEN,or any combinations thereof. In other embodiments, the procoagulantcompound comprises a clotting factor or fragment thereof, a syntheticprocoagulant polypeptide, and a PEG heterologous moiety, wherein theprocoagulant compound further comprises a heterologous moiety selectedfrom an immunoglobulin constant region or portion thereof (e.g., an Fcregion), a PAS sequence, HES, and albumin, fragment, or variant thereof,an XTEN, or any combinations thereof.

In other embodiments, the procoagulant compound comprises two syntheticprocoagulant peptides and a PEG heterologous moiety, wherein theprocoagulant compound further comprises a heterologous moiety selectedfrom an immunoglobulin constant region or portion thereof (e.g., an Fcregion), a PAS sequence, HES, and albumin, fragment, or variant thereof,an XTEN, or any combinations thereof. In yet another embodiment, theprocoagulant compound comprises a clotting factor or fragment thereof, aclotting factor cofactor (e.g., Factor Va if the clotting factor inFactor X; or Tissue Factor if the clotting factor is Factor VII), and aPEG heterologous moiety, wherein the procoagulant compound furthercomprises a heterologous moiety selected from an immunoglobulin constantregion or portion thereof (e.g., an Fc region), a PAS sequence, HES, andalbumin, fragment, or variant thereof, an XTEN, or any combinationsthereof.

Also provided by the invention are procoagulant compounds of theinvention comprising heterologous moieties which can provide additionaladvantages such as increased solubility, stability and circulating timeof the polypeptide, or decreased immunogenicity (see U.S. Pat. No.4,179,337). Such heterologous moieties for modification can be selectedfrom water soluble polymers including, but not limited to, polyethyleneglycol, ethylene glycol/propylene glycol copolymers,carboxymethylcellulose, dextran, polyvinyl alcohol, or any combinationsthereof.

The polymer can be of any molecular weight, and can be branched orunbranched. For polyethylene glycol, in one embodiment, the molecularweight is between about 1 kDa and about 100 kDa for ease in handling andmanufacturing. Other sizes can be used, depending on the desired profile(e.g., the duration of sustained release desired, the effects, if any onbiological activity, the ease in handling, the degree or lack ofantigenicity and other known effects of the polyethylene glycol to aprotein or analog). For example, the polyethylene glycol can have anaverage molecular weight of about 200, 500, 1000, 1500, 2000, 2500,3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500,9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000,13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500,18,000, 18,500, 19,000, 19,500, 20,000, 25,000, 30,000, 35,000, 40,000,45,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000,90,000, 95,000, or 100,000 kDa.

In some embodiments, the polyethylene glycol can have a branchedstructure. Branched polyethylene glycols are described, for example, inU.S. Pat. No. 5,643,575; Morpurgo et al., Appl. Biochem. Biotechnol.56:59-72 (1996); Vorobjev et al., Nucleosides Nucleotides 18:2745-2750(1999); and Caliceti et al., Bioconjug. Chem. 10:638-646 (1999), each ofwhich is incorporated herein by reference in its entirety.

The number of polyethylene glycol moieties attached to each procoagulantcompound of the invention (i.e., the degree of substitution) can alsovary. For example, the PEGylated procoagulant compound can be linked, onaverage, to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, or morepolyethylene glycol molecules. Similarly, the average degree ofsubstitution within ranges such as 1-3, 2-4, 3-5, 4-6, 5-7, 6-8, 7-9,8-10, 9-11, 10-12, 11-13, 12-14, 13-15, 14-16, 15-17, 16-18, 17-19, or18-20 polyethylene glycol moieties per protein molecule. Methods fordetermining the degree of substitution are discussed, for example, inDelgado et al., Crit. Rev. Thera. Drug Carrier Sys. 9:249-304 (1992).

In some embodiments, the procoagulant compound can be PEGylated. APEGylated procoagulant compound comprises at least one polyethyleneglycol (PEG) molecule. In other embodiments, the polymer can bewater-soluble. Non-limiting examples of the polymer can be poly(alkyleneoxide), poly(vinyl pyrrolidone), poly(vinyl alcohol), polyoxazoline, orpoly(acryloylmorpholine). Additional types of polymer-conjugation toclotting factors are disclosed in U.S. Pat. No. 7,199,223. See also,Singh et al. Curr. Med. Chem. 15:1802-1826 (2008).

(k) Hydroxyethyl Starch (HES)

In certain embodiments, at least one heterologous moiety is a polymer,e.g., hydroxyethyl starch (HES) or a derivative thereof. Hydroxyethylstarch (HES) is a derivative of naturally occurring amylopectin and isdegraded by alpha-amylase in the body. HES is a substituted derivativeof the carbohydrate polymer amylopectin, which is present in corn starchat a concentration of up to 95% by weight. HES exhibits advantageousbiological properties and is used as a blood volume replacement agentand in hemodilution therapy in the clinics (Sommermeyer et al.,Krankenhauspharmazie, 8(8), 271-278 (1987); and Weidler et al.,Arzneim.-Forschung/Drug Res., 41, 494-498 (1991)).

Amylopectin contains glucose moieties, wherein in the main chainalpha-1,4-glycosidic bonds are present and at the branching sitesalpha-1,6-glycosidic bonds are found. The physical-chemical propertiesof this molecule are mainly determined by the type of glycosidic bonds.Due to the nicked alpha-1,4-glycosidic bond, helical structures withabout six glucose-monomers per turn are produced. The physico-chemicalas well as the biochemical properties of the polymer can be modified viasubstitution. The introduction of a hydroxyethyl group can be achievedvia alkaline hydroxyethylation. By adapting the reaction conditions itis possible to exploit the different reactivity of the respectivehydroxy group in the unsubstituted glucose monomer with respect to ahydroxyethylation. Owing to this fact, the skilled person is able toinfluence the substitution pattern to a limited extent.

HES is mainly characterized by the molecular weight distribution and thedegree of substitution. The degree of substitution, denoted as DS,relates to the molar substitution, is known to the skilled people. SeeSommermeyer et al., Krankenhauspharmazie, 8(8), 271-278 (1987), as citedabove, in particular p. 273.

In one embodiment, hydroxyethyl starch has a mean molecular weight(weight mean) of from 1 to 300 kD, from 2 to 200 kD, from 3 to 100 kD,or from 4 to 70 kD. hydroxyethyl starch can further exhibit a molardegree of substitution of from 0.1 to 3, preferably 0.1 to 2, morepreferred, 0.1 to 0.9, preferably 0.1 to 0.8, and a ratio between C2:C6substitution in the range of from 2 to 20 with respect to thehydroxyethyl groups. A non-limiting example of HES having a meanmolecular weight of about 130 kD is a HES with a degree of substitutionof 0.2 to 0.8 such as 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, or 0.8, preferablyof 0.4 to 0.7 such as 0.4, 0.5, 0.6, or 0.7.

In a specific embodiment, HES with a mean molecular weight of about 130kD is VOLUVEN® from Fresenius. VOLUVEN® is an artificial colloid,employed, e.g., for volume replacement used in the therapeuticindication for therapy and prophylaxis of hypovolemia. Thecharacteristics of VOLUVEN® are a mean molecular weight of130,000+/−20,000 D, a molar substitution of 0.4 and a C2:C6 ratio ofabout 9:1. In other embodiments, ranges of the mean molecular weight ofhydroxyethyl starch are, e.g., 4 to 70 kD or 10 to 70 kD or 12 to 70 kDor 18 to 70 kD or 50 to 70 kD or 4 to 50 kD or 10 to 50 kD or 12 to 50kD or 18 to 50 kD or 4 to 18 kD or 10 to 18 kD or 12 to 18 kD or 4 to 12kD or 10 to 12 kD or 4 to 10 kD.

In still other embodiments, the mean molecular weight of hydroxyethylstarch employed is in the range of from more than 4 kD and below 70 kD,such as about 10 kD, or in the range of from 9 to 10 kD or from 10 to 11kD or from 9 to 11 kD, or about 12 kD, or in the range of from 11 to 12kD) or from 12 to 13 kD or from 11 to 13 kD, or about 18 kD, or in therange of from 17 to 18 kD or from 18 to 19 kD or from 17 to 19 kD, orabout 30 kD, or in the range of from 29 to 30, or from 30 to 31 kD, orabout 50 kD, or in the range of from 49 to 50 kD or from 50 to 51 kD orfrom 49 to 51 kD.

In certain embodiments, the heterologous moiety can be a mixture ofhydroxyethyl starches having different mean molecular weights and/ordifferent degrees of substitution and/or different ratios of C2:C6substitution. Therefore, mixtures of hydroxyethyl starches can beemployed having different mean molecular weights and different degreesof substitution and different ratios of C2:C6 substitution, or havingdifferent mean molecular weights and different degrees of substitutionand the same or about the same ratio of C2:C6 substitution, or havingdifferent mean molecular weights and the same or about the same degreeof substitution and different ratios of C2:C6 substitution, or havingthe same or about the same mean molecular weight and different degreesof substitution and different ratios of C2:C6 substitution, or havingdifferent mean molecular weights and the same or about the same degreeof substitution and the same or about the same ratio of C2:C6substitution, or having the same or about the same mean molecularweights and different degrees of substitution and the same or about thesame ratio of C2:C6 substitution, or having the same or about the samemean molecular weight and the same or about the same degree ofsubstitution and different ratios of C2:C6 substitution, or having aboutthe same mean molecular weight and about the same degree of substitutionand about the same ratio of C2:C6 substitution.

(l) Polysialic Acids (PSA)

In certain embodiments, at least one heterologous moiety is a polymer,e.g., polysialic acids (PSAs) or a derivative thereof. Polysialic acids(PSAs) are naturally occurring unbranched polymers of sialic acidproduced by certain bacterial strains and in mammals in certain cellsRoth J., et al. (1993) in Polysialic Acid: From Microbes to Man, edsRoth J., Rutishauser U., Troy F. A. (Birkhäuser Verlag, Basel,Switzerland), pp 335-348. They can be produced in various degrees ofpolymerisation from n=about 80 or more sialic acid residues down to n=2by limited acid hydrolysis or by digestion with neuraminidases, or byfractionation of the natural, bacterially derived forms of the polymer.

The composition of different polysialic acids also varies such thatthere are homopolymeric forms i.e. the alpha-2,8-linked polysialic acidcomprising the capsular polysaccharide of E. coli strain K1 and thegroup-B meningococci, which is also found on the embryonic form of theneuronal cell adhesion molecule (N-CAM). Heteropolymeric forms alsoexist—such as the alternating alpha-2,8 alpha-2,9 polysialic acid of E.coli strain K92 and group C polysaccharides of N. meningitidis. Sialicacid can also be found in alternating copolymers with monomers otherthan sialic acid such as group W135 or group Y of N. meningitidis.Polysialic acids have important biological functions including theevasion of the immune and complement systems by pathogenic bacteria andthe regulation of glial adhesiveness of immature neurons during foetaldevelopment (wherein the polymer has an anti-adhesive function) Cho andTroy, P.N.A.S., USA, 91 (1994) 11427-11431, although there are no knownreceptors for polysialic acids in mammals.

The alpha-2,8-linked polysialic acid of E. coli strain K1 is also knownas ‘colominic acid’ and is used (in various lengths) to exemplify thepresent invention. Various methods of attaching or conjugatingpolysialic acids to a polypeptide have been described (for example, seeU.S. Pat. No. 5,846,951; WO-A-0187922, and US 2007/0191597 A1, which areincorporated herein by reference in their entireties.

(m) Clearance Receptors

In certain aspects, the in vivo half-life of a therapeutic polypeptidein a procoagulant compound of the invention can be extended where theprocoagulant compound comprises at least one heterologous moleculecomprising a clearance receptor, fragment, variant, or derivativethereof. In specific aspects wherein the therapeutic peptide is FactorVIII, Factor IX or Factor X, soluble forms of clearance receptors, suchas the low density lipoprotein-related protein receptor LRP1, orfragments thereof, can block binding of Factor VIII, Factor IX or FactorX to clearance receptors and thereby extend its in vivo half-life.

LRP1 is a 600 kDa integral membrane protein that is implicated in thereceptor-mediate clearance of a variety of proteins, such as FactorVIII. See, e.g., Lenting et al., Haemophilia 16:6-16 (2010). LRP1 alsomediates clearance of Factor Xa (see, e.g., Narita et al., Blood91:555-560 (1998)) and Factor IX (see, e.g., Strickland & Medved. J.Thromb. Haemostat. 4:1484-1486 (2006).

Other suitable FVIII clearance receptors are, e.g., LDLR (low-densitylipoprotein receptor), VLDLR (very low-density lipoprotein receptor),and megalin (LRP-2), or fragments thereof. See, e.g.,Bovenschen et al.,Blood 106:906-912 (2005); Bovenschen, Blood 116:5439-5440 (2010);Martinelli et al., Blood 116:5688-5697 (2010).

2. Visualization and Location

In certain embodiments, a heterologous moiety facilitates visualizationor localization of the procoagulant compounds of the invention. Myriadpeptides and other moieties for insertion or conjugation into a compoundwhich facilitate visualization or localization are known in the art.Such moieties can be used to facilitate visualization or localization invitro, in vivo, ex vivo or any combination thereof.

Since thrombin plays a central role in the coagulation cascade,detection of imaging of its activity in vivo is highly desired.Accordingly, various heterologous moiety facilitates visualization orlocalization of the procoagulant compounds of the invention (e.g.,fluorescent dyes) can be engineered into the procoagulant compounds ofthe invention. In some embodiments, fluorescent dyes can be engineeredto be non-fluorescent until their amines are regenerated upon thrombincleavage.

Non-limiting examples of peptides or polypeptides which enablevisualization or localization include biotin acceptor peptides which canfacilitate conjugation of avidin- and streptavidin-based reagents,lipoic acid acceptor peptides which can facilitate conjugation ofthiol-reactive probes to bound lipoic acid or direct ligation offluorescent lipoic acid analogs, fluorescent proteins, e.g., greenfluorescent protein (GFP) and variants thereof (e.g., EGFP, YFP such asEYFP, mVenus, YPet or Citrine, or CFP such as Cerulean or ECFP) or redfluorescent protein (RFP), cysteine-containing peptides for ligation ofbiarsenical dyes such as 4′,5′-bis(1,3,2-dithioarsolan-2-yl)fluorescein(FlAsH), or for conjugating metastable technetium, peptides forconjugating europium clathrates for fluorescence resonance energytransfer (FRET)-based proximity assays, any variants, thereof, and anycombination thereof.

Procoagulant compounds of the present disclosure labeled by thesetechniques can be used, for example, for 3-D imaging of pathologicalthrombus formation and dissolution, tumor imaging in procoagulantmalignancies, flow cytometric quantitation and characterization ofprocoagulant microparticles in blood and plasma, monitoring of thrombusformation by intravital microscopy.

3. Targeting Moieties, Anchors and Other Moieties

In some embodiments, procoagulant compounds of the invention cancomprise a heterologous moiety that targets the compound to specificlocation, e.g., to platelets to enhance the efficacy of the compound bylocalizing the clotting factor or procoagulation peptide of the compoundto the site of coagulation (a “targeting moiety”). In some embodiment,the targeting moiety binds to a target molecule expressed on platelets.Preferably the targeted molecules are not expressed on cells or tissuesother than platelets, i.e., the targeting moieties specifically bind toplatelets.

In one embodiment, receptors/conformations found on resting plateletsare targeted. By doing so, sites for coagulation could be primed forenhanced efficacy. Targeting such molecule can also extend half life ofthe clotting factor and/or prevent clearance. Examples of such targetsinclude GpIb of the GpIb/V/IX complex, and GpVI and nonactive form ofGPIIb/IIIa. See, e.g., Schwarz et al. Circulation Research. 99:25-33(2006); U.S. Pat. Publ. 20070218067; Peterson et al. Hemostasis,Thrombosis, and Vascular Biology 101:937 (2003); WO 2010115866; Lin etal. Journal of Thrombosis and Haemostasis 8:1773 (2010).

The procoagulant compound of the invention can comprise one or more thanone targeting moiety. In some embodiments, two or more targetingmoieties can be linked to each other (e.g., via a linker). When two ormore targeting moieties are present in a procoagulant compound of theinvention, the targeting moieties can be the same or different.

In one embodiment, a targeting moiety is fused to a procoagulantcompound of the invention by a protease cleavable linker which can becleaved to remove the targeting moiety at the site of a clot. In anotherembodiment, a targeting moiety is not attached via a cleavable linkerand, therefore, is not cleaved at the site of a clot.

In one embodiment, the targeting moiety is located on the N- orC-terminus of the procoagulant compound. In one embodiment, a targetingmoiety is not genetically fused directly to a procoagulant compound ofthe invention, but rather is chemically linked via a linker or achemical bond to the construct (see, e.g., U.S. Pat. No. 7,381,408).

In one embodiment, a procoagulant compound of the invention comprises atleast an antigen binding site (e.g., an antigen binding site of anantibody, antibody variant, or antibody fragment), a polypeptide, areceptor binding portion of ligand, or a ligand binding portion of areceptor which specifically binds to platelets, e.g., resting oractivated platelets. Exemplary targeting moieties include scFv moleculesor peptides which bind to molecules to be targeted.

In some embodiments, a procoagulant compound of the invention comprisesan anchor or scaffolding molecule, e.g., a lipid, a carbohydrate, or asulfhydryl group. For example, a sulfhydryl group in an N-terminalcysteine can be used to anchor a procoagulant compound of the inventionto another molecule, cell, or other surface. For example, a lipid anchorcan be used to anchor a procoagulant compound of the invention to a cellsurface or a lipid bilayer (e.g., a liposome).

In some embodiments, a procoagulant compound of the invention cancomprise a heterogous molecule comprising a non-peptidic active agentuseful for the treatment of bleeding disorders. In some embodiments, thenon-peptidic active agent is a procoagulant molecule. In someembodiments, the non-peptidic active agent is a small molecule drug.

III. Pharmaceutical Compositions

The invention also provides pharmaceutical compositions containing atleast one procoagulant compound of the present and a pharmaceuticallyacceptable carrier.

The term “pharmaceutically acceptable carrier” means allpharmaceutically acceptable ingredients known to those of skill in theart, which are typically considered non-active ingredients. The term“pharmaceutically acceptable carrier” includes, e.g., solvents, solid orliquid diluents, additives, vehicles, adjuvants, excipients, glidants,binders, granulating agents, dispersing agents, suspending agents,wetting agents, lubricating agents, disintegrants, solubilizers,stabilizers, preservatives, emulsifiers, fillers, preservatives (e.g.,anti-oxidants), flavoring agents, sweetening agents, thickening agents,buffering agents, coloring agents and the like, as well as any mixturesthereof. Exemplary carriers (i.e., excipients) are described in, e.g.,Handbook of Pharmaceutical Manufacturing Formulations, Volumes 1-6,Niazi, Sarfaraz K., Taylor & Francis Group 2005, which is incorporatedherein by reference in its entirety.

Pharmaceutical compositions can additionally comprise, for example, oneor more of water, buffers (e.g., neutral buffered saline or phosphatebuffered saline), ethanol, mineral oil, vegetable oil,dimethylsulfoxide, carbohydrates (e.g., glucose, mannose, sucrose ordextrans), mannitol, proteins, adjuvants, polypeptides or amino acidssuch as glycine, antioxidants, chelating agents such as EDTA orglutathione and/or preservatives.

Pharmaceutical compositions can be formulated for any appropriate mannerof administration, including, for example, topical (e.g., transdermal orocular), oral, buccal, nasal, vaginal, rectal or parenteraladministration.

The term parenteral as used herein includes subcutaneous, intradermal,intravascular (e.g., intravenous), intramuscular, spinal, intracranial,intrathecal, intraocular, periocular, intraorbital, intrasynovial andintraperitoneal injection, as well as any similar injection or infusiontechnique. It is preferred that subcutaneous, intraperitoneal, buccal,intravenous and other parenteral formulations are sterile and endotoxinfree. Procoagulant compounds of the present disclosure can beadministered parenterally in a sterile medium.

The procoagulant compound, depending on the vehicle and concentrationused, can either be suspended or dissolved in the vehicle. In oneembodiment, adjuvants such as local anesthetics, preservatives andbuffering agents can be dissolved in the vehicle. In one example, theprocoagulant compounds of the present invention are administered to thesubject using a non-intravenous route, e.g., by subcutaneous, nasal,buccal, oral or pulmonary delivery.

Forms suitable for oral use include, for example, tablets, troches,lozenges, aqueous or oily suspensions, dispersible powders or granules,emulsion, hard or soft capsules, or syrups or elixirs. Compositionsprovided herein can be formulated as a lyophilizate.

The pharmaceutical composition can be also for example a suspension,emulsion, sustained release formulation, cream, gel or powder. Thepharmaceutical composition can be formulated as a suppository, withtraditional binders and carriers such as triglycerides.

In one example, the pharmaceutical composition is a liquid formulation,e.g., a buffered, isotonic, aqueous solution. In one example, thepharmaceutical composition has a pH that is physiologic, or close tophysiologic. In another example, the aqueous formulation has aphysiologic or close to physiologic osmolarity and salinity. It cancontain sodium chloride and/or sodium acetate.

Pharmaceutical compositions intended for oral use can be preparedaccording to any method known for the manufacture of pharmaceuticalcompositions. Such pharmaceutical compositions can contain one or moreagents chosen from the group consisting of sweetening agents, flavoringagents, coloring agents and preservative agents in order to providepharmaceutically elegant and palatable preparations. Tablets can containthe active ingredient in admixture with non-toxic pharmaceuticallyacceptable excipients that are suitable for the manufacture of tablets.These excipients can be for example, inert diluents, such as calciumcarbonate, sodium carbonate, lactose, calcium phosphate or sodiumphosphate; granulating and disintegrating agents, for example, cornstarch, or alginic acid; binding agents, for example starch, gelatin oracacia, and lubricating agents, for example magnesium stearate, stearicacid or talc. The tablets can be uncoated or they can be coated by knowntechniques. In some cases such coatings can be prepared by knowntechniques to delay disintegration and absorption in thegastrointestinal tract and thereby provide a sustained action over alonger period (i.e., tablets can be enterically coated). For example, atime delay material such as glyceryl monosterate or glyceryl distearatecan be employed.

Pharmaceutical compositions for oral use can also be presented as hardgelatin capsules, wherein the active ingredient is mixed with an inertsolid diluent, for example, calcium carbonate, calcium phosphate orkaolin, or as soft gelatin capsules wherein the active ingredient ismixed with water or an oil medium, for example peanut oil, liquidparaffin or olive oil. In another example, the active ingredient isformulated in capsules containing optionally coated microtablets ormicropellets. Pharmaceutical compositions for oral use can also bepresented as lozenges.

Aqueous suspensions contain the active ingredient(s) in admixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients include suspending agents (e.g., sodiumcarboxymethylcellulose, methylcellulose, hydropropyl methylcellulose,sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia);and dispersing or wetting agents (e.g., naturally-occurring phosphatidessuch as lecithin, condensation products of an alkylene oxide with fattyacids such as polyoxyethylene stearate, condensation products ofethylene oxide with long chain aliphatic alcohols such asheptadecaethyleneoxycetanol, condensation products of ethylene oxidewith partial esters derived from fatty acids and a hexitol such aspolyoxyethylene sorbitol monooleate, or condensation products ofethylene oxide with partial esters derived from fatty acids and hexitolanhydrides such as polyethylene sorbitan monooleate). Aqueoussuspensions can also comprise one or more preservatives, for exampleethyl, or n-propyl p-hydroxybenzoate, one or more coloring agents, oneor more flavoring agents, and one or more sweetening agents, such assucrose or saccharin.

Oily suspensions can be formulated by suspending the active ingredientsin a vegetable oil, for example arachis oil, olive oil, sesame oil orcoconut oil, or in a mineral oil such as liquid paraffin. The oilysuspensions can contain a thickening agent, for example beeswax, hardparaffin or cetyl alcohol. Sweetening agents and flavoring agents can beadded to provide palatable oral preparations. These compositions can bepreserved by the addition of an anti-oxidant such as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water can provide the active ingredient inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents orsuspending agents are exemplified by those already mentioned above.Additional excipients, for example sweetening, flavoring and coloringagents, can also be present.

Pharmaceutical compositions of the present disclosure can also be in theform of oil-in-water emulsions. The oily phase can be a vegetable oil ora mineral oil or mixtures of these. Suitable emulsifying agents can benaturally-occurring gums, for example gum acacia or gum tragacanth,naturally-occurring phosphatides, for example soy bean, lecithin, andesters or partial esters derived from fatty acids and hexitol,anhydrides, for example sorbitan monooleate, and condensation productsof the said partial esters with ethylene oxide, for examplepolyoxyethylene sorbitan monooleate. The emulsions can also containsweetening and flavoring agents.

Syrups and elixirs can be formulated with sweetening agents, for exampleglycerol, propylene glycol, sorbitol, glucose or sucrose. Suchformulations can also contain a demulcent, a preservative, a flavoringagent or a coloring agent. The pharmaceutical compositions can be in theform of a sterile injectable aqueous or oleaginous suspension. Thissuspension can be formulated according to the known art using thosesuitable dispersing or wetting agents and suspending agents that havebeen mentioned above. The sterile injectable preparation can also be asterile injectable solution or suspension in a non-toxic parentallyacceptable diluent or solvent, for example as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that can beemployed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose any bland fixed oilcan be employed including synthetic mono- or diglycerides. In addition,fatty acids such as oleic acid find use in the preparation ofinjectables.

The procoagulant compounds of the present disclosure can also beadministered in the form of suppositories, e.g., for rectaladministration of the drug. These pharmaceutical compositions can beprepared by mixing the drug with a suitable non-irritating excipientthat is solid at ordinary temperatures but liquid at the rectaltemperature and will therefore melt in the rectum to release the drug.Such materials include cocoa butter and poly ethylene glycols.

Procoagulant compounds of the present disclosure can be formulated forlocal or topical administration, such as for topical application to theskin, wounds or mucous membranes, such as in the eye. Formulations fortopical administration typically comprise a topical vehicle combinedwith active agent(s), with or without additional optional components.Suitable topical vehicles and additional components are well known inthe art, and it will be apparent that the choice of a vehicle willdepend on the particular physical form and mode of delivery. Topicalvehicles include water; organic solvents such as alcohols (e.g., ethanolor isopropyl alcohol) or glycerin; glycols (e.g., butylene, isoprene orpropylene glycol); aliphatic alcohols (e.g., lanolin); mixtures of waterand organic solvents and mixtures of organic solvents such as alcoholand glycerin; lipid-based materials such as fatty acids, acylglycerols(including oils, such as mineral oil, and fats of natural or syntheticorigin), phosphoglycerides, sphingolipids and waxes; protein-basedmaterials such as collagen and gelatin; silicone-based materials (bothnon-volatile and volatile); and hydrocarbon-based materials such asmicrosponges and polymer matrices.

A composition can further include one or more components adapted toimprove the stability or effectiveness of the applied formulation, suchas stabilizing agents, suspending agents, emulsifying agents, viscosityadjusters, gelling agents, preservatives, antioxidants, skin penetrationenhancers, moisturizers and sustained release materials. Examples ofsuch components are described in Martindale—The Extra Pharmacopoeia(Pharmaceutical Press, London 1993) and Martin (ed.), Remington'sPharmaceutical Sciences. Formulations can comprise microcapsules, suchas hydroxymethylcellulose or gelatin-microcapsules, liposomes, albuminmicrospheres, microemulsions, nanoparticles or nanocapsules.

Pharmaceutical compositions suitable for topical administration to theeye also include eye drops wherein the active ingredients are dissolvedor suspended in suitable carrier, especially an aqueous solvent for theactive ingredients. The anti-inflammatory active ingredients may, forexample, be present in such formulations in a concentration of 0.5 to20%, such as 0.5 to 10%, for example about 1.5% w/w. For therapeuticpurposes, the active compounds of the present disclosure are ordinarilycombined with one or more adjuvants appropriate to the indicated routeof administration. The compounds can be admixed with lactose, sucrose,starch powder, cellulose esters of alkanoic acids, cellulose alkylesters, talc, stearic acid, magnesium stearate, magnesium oxide, sodiumand calcium salts of phosphoric and sulfuric acids, gelatin, acacia gum,sodium alginate, polyvinylpyrrolidone, and/or polyvinyl alcohol, andthen tableted or encapsulated for convenient administration. Suchcapsules or tablets can contain a controlled-release formulation as canbe provided in a dispersion of active compound in hydroxypropylmethylcellulose.

Formulations for parenteral administration can be in the form of aqueousor non-aqueous isotonic sterile injection solutions or suspensions.These solutions and suspensions can be prepared from sterile powders orgranules having one or more of the carriers or diluents mentioned foruse in the formulations for oral administration. The compounds can bedissolved in water, polyethylene glycol, propylene glycol, ethanol, cornoil, cottonseed oil, peanut oil, sesame oil, benzyl alcohol, sodiumchloride, and/or various buffers. Other adjuvants and modes ofadministration are well and widely known in the pharmaceutical art.

Alternatively, the active ingredients can be formulated in a cream withan oil-in-water cream base. If desired, the aqueous phase of the creambase can include, for example at least 30% w/w of a polyhydric alcoholsuch as propylene glycol, butane-1,3-diol, mannitol, sorbitol, glycerol,polyethylene glycol and mixtures thereof. The topical formulation candesirably include a compound, which enhances absorption or penetrationof the active ingredient through the skin or other affected areas.Examples of such dermal penetration enhancers include dimethylsulfoxideand related analogs. The compounds of this present disclosure can alsobe administered by a transdermal device. In one embodiment, topicaladministration will be accomplished using a patch either of thereservoir and porous membrane type or of a solid matrix variety. Ineither case, the active agent is delivered continuously from thereservoir or microcapsules through a membrane into the active agentpermeable adhesive, which is in contact with the skin or mucosa of therecipient.

If the active agent is absorbed through the skin, a controlled andpredetermined flow of the active agent is administered to the recipient.In the case of microcapsules, the encapsulating agent can also functionas the membrane. The transdermal patch can include the compound in asuitable solvent system with an adhesive system, such as an acrylicemulsion, and a polyester patch. The oily phase of the emulsions of thispresent disclosure can be constituted from known ingredients in a knownmanner. While the phase can comprise merely an emulsifier, it cancomprise a mixture of at least one emulsifier with a fat or oil or withboth a fat and an oil. In one embodiment, a hydrophilic emulsifier isincluded together with a lipophilic emulsifier, which acts as astabilizer. The phase may, for example, include both an oil and a fat.Together, the emulsifier(s) with or without stabilizer(s) make-up theso-called emulsifying wax, and the wax together with the oil and fatmake up the so-called emulsifying ointment base, which forms the oily,dispersed phase of the cream formulations.

Emulsifiers and emulsion stabilizers suitable for use in the formulationof the present disclosure include Tween 60, Span 80, cetostearylalcohol, myristyl alcohol, glyceryl monostearate, and sodium laurylsulfate, among others. The choice of suitable oils or fats for theformulation is based on achieving the desired cosmetic properties, sincethe solubility of the active compound in most oils likely to be used inpharmaceutical emulsion formulations is very low. Thus, the cream may,for example, be a non-greasy, non-staining and washable product withsuitable consistency to avoid leakage from tubes or other containers.Straight or branched chain, mono- or dibasic alkyl esters such asdi-isoadipate, isocetyl stearate, propylene glycol diester of coconutfatty acids, isopropyl myristate, decyl oleate, isopropyl palmitate,butyl stearate, 2-ethylhexyl palmitate or a blend of branched chainesters can be used. These can be used alone or in combination dependingon the properties required. Alternatively, high melting point lipidssuch as white soft paraffin and/or liquid paraffin or other mineral oilscan be used.

A pharmaceutical composition can be formulated as inhaled formulations,including sprays, mists, or aerosols. For inhalation formulations, thecompounds provided herein can be delivered via any inhalation methodsknown to those skilled in the art. Such inhalation methods and devicesinclude, but are not limited to, metered dose inhalers with propellantssuch as CFC or HFA or propellants that are physiologically andenvironmentally acceptable. Other suitable devices are breath operatedinhalers, multidose dry powder inhalers and aerosol nebulizers. Aerosolformulations for use in the subject method typically includepropellants, surfactants and co-solvents and can be filled intoconventional aerosol containers that are closed by a suitable meteringvalve.

Formulations suitable for inhalation or insufflation include solutionsand suspensions in pharmaceutically acceptable aqueous or organicsolvents, or mixtures thereof, and powders. The liquid or solidcompositions can contain suitable pharmaceutically acceptable excipientsas describe above. The compositions can be administered by oral or nasalrespiratory route for local or systemic effect. Compositions can benebulized by use of inert gases or vaporized, and breathed directly fromthe nebulizing/vaporizing device or the nebulizing device can beattached to a facemask tent or intermittent positive pressure-breathingmachine.

Inhalant compositions can comprise liquid or powdered compositionscontaining the active ingredient that are suitable for nebulization andintrabronchial use, or aerosol compositions administered via an aerosolunit dispensing metered doses. Suitable liquid compositions comprise theactive ingredient in an aqueous, pharmaceutically acceptable inhalantsolvent, e.g., isotonic saline or bacteriostatic water. The solutionsare administered by means of a pump or squeeze-actuated nebulized spraydispenser, or by any other conventional means for causing or enablingthe requisite dosage amount of the liquid composition to be inhaled intothe patient's lungs. Suitable formulations, wherein the carrier is aliquid, for administration, as for example, a nasal spray or as nasaldrops, include aqueous or oily solutions of the active ingredient.

Formulations or compositions suitable for nasal administration, whereinthe carrier is a solid, include a coarse powder having a particle size,for example, in the range of 20 to 500 microns which is administered inthe manner in which snuff is administered (i.e., by rapid inhalationthrough the nasal passage from a container of the powder held close upto the nose). Suitable powder compositions include, by way ofillustration, powdered preparations of the active ingredient thoroughlyintermixed with lactose or other inert powders acceptable forintrabronchial administration. The powder compositions can beadministered via an aerosol dispenser or encased in a breakable capsulewhich can be inserted by the patient into a device that punctures thecapsule and blows the powder out in a steady stream suitable forinhalation.

Pharmaceutical compositions can be formulated as sustained releaseformulations (i.e., a formulation such as a capsule that effects a slowrelease of modulator following administration). Such formulations cangenerally be prepared using well known technology and administered by,for example, oral, rectal or subcutaneous implantation, or byimplantation at the desired target site. Carriers for use within suchformulations are biocompatible, and can also be biodegradable;preferably the formulation provides a relatively constant level ofmodulator release. The amount of modulator contained within a sustainedrelease formulation depends upon, for example, the site of implantation,the rate and expected duration of release and the nature of thecondition to be treated or prevented.

In one example, the pharmaceutical formulations provided herein caninclude one or more additional active agent (i.e., other biologicallyactive ingredient). In one example, the additional active agent isselected from known drugs approved for the treatment of a coagulationdisorder, such as hemophilia A. For example, the pharmaceuticalformulation can further include a blood coagulation factor.

Pharmaceutical compositions can be formulated with an agent to improvebioavailability, such an as organic solvent. For example, Cremophor EL®(Product No. 00647/1/63; BASF Aktiengesellschaft, Germany) is apolyethoxylated castor oil which is prepared by reacting 35 moles ofethylene oxide with each mole of castor oil. It can be used to stabilizeemulsions of non-polar materials in aqueous systems. Alternatively,peptide, peptide derivative or dual peptide can be incorporated withinor bound to a proteinaceous micro or nano-particle for improvedbioavailability.

Suitable micro- and nano-particles are described in U.S. Pat. No.5,439,686 (Desai et al; Vivorx Pharmaceuticals, Inc., CA) and U.S. Pat.No. 5,498,421 (Grinstaff et al; Vivorx Pharmaceuticals, Inc., CA).Suitably, the proteinaceous nano-particle comprises human serum albumin,particularly human serum albumin or a recombinant form thereof. WO2007/077561 (Gabbai; Do-Coop Technologies Ltd., Israel) describe anothersuitable carrier comprising nanostructures and a liquid, referred totherein as NEOWATER™.

For veterinary use, a compound of the present disclosure is administeredas a suitably acceptable formulation in accordance with normalveterinary practice and the veterinary surgeon will determine the dosingregimen and route of administration which will be most appropriate for aparticular animal. For administration to non-human animals, thecomposition can be added to the animal feed or drinking water. It can beconvenient to formulate the animal feed and drinking water compositionsso that the animal takes in a therapeutically appropriate quantity ofthe composition along with its diet. It can also be convenient topresent the composition as a premix for addition to the feed or drinkingwater.

Dosage levels of the order of from about 0.005 mg to about 80 mg perkilogram of body weight per day are useful in the treatment of thediseases and conditions described herein (e.g., about 0.35 mg to about5.6 g per human patient per day, based on an average adult person weightof 70 kg). The amount of active ingredient that can be combined with thecarrier materials to produce a single dosage form will vary dependingupon the host treated and the particular mode of administration. Dosageunit forms will generally contain between from about 1 mg to about 500mg of an active ingredient. The daily dose can be administered in one tofour doses per day. In the case of skin conditions, it may, for example,be applied as a topical preparation of compounds of this presentdisclosure on the affected area one to four times a day.

It will be understood, however, that the specific dose level for anyparticular patient will depend upon a variety of factors including theactivity of the specific compound employed, the age, body weight,general health, sex, diet, time of administration, route ofadministration, and rate of excretion, drug combination and the severityof the particular disease undergoing therapy.

IV. Methods of Making

The procoagulant compounds of the present disclosure can be produced bychemical synthesis, recombinant DNA technology, biochemical or enzymaticfragmentation of larger molecules, combinations of the foregoing or byany other method.

In one example, the method comprises forming the amino acid sequence ofthe compound, or a retro-, inverso- or retro-inverso variant thereofusing solid-phase peptide synthesis. Exemplary methods of makingprocoagulant compounds of the invention are described herein inExample 1. Other methods to form synthetic peptides are known to thoseof skill in the art.

For example, the procoagulant compounds of the present disclosure can besynthesized using solid-phase peptide synthesis as described in “FmocSolid Phase Peptide Synthesis—A Practical Approach”, edited by W. C.Chan, P. D. White, Oxford University Press, New York 2000 and referencestherein. Temporary N-amino group protection is afforded, e.g., by a9-fluorenylmethyloxycarbonyl (Fmoc) group. Repetitive cleavage of thishighly base-labile protecting group is effected, e.g., using 20%piperidine in N,N-dimethylformamide. Side-chain functionalities can beprotected as their butyl ethers (in the case of serine, threonine andtyrosine), butyl esters (in the case of glutamic acid and asparticacid), butyloxycarbonyl derivative (in the case of lysine andhistidine), trityl derivative (in the case of cysteine, asparagine andglutamine) and 4-methoxy-2,3,6-trimethylbenzenesulphonyl derivative (inthe case of arginine).

The solid-phase support can be based on a polydimethyl-acrylamidepolymer constituted from the three monomers dimethylacrylamide(backbone-monomer), bisacryloylethylene diamine (cross linker) andacryloylsarcosine methyl ester (functionalising agent), or can be basedon polyethylene glycol (PEG), such as Rink Amide resin (e.g., NovaPEGRink Amide). The peptide-to-resin cleavable linked agent can be theacid-labile 4-hydroxymethyl-phenoxyacetic acid derivative, or in case ofC-terminal amides, the Rink-amide linker. All amino acid derivatives canbe added as their preformed symmetrical anhydride derivatives with theexception of asparagine and glutamine, which are added using a reversedN,N-dicyclohexyl-carbodiimide/1-hydroxybenzotriazole mediated couplingprocedure.

Alternatively, other peptide coupling reagents, such asO-benzotriazole-N,N,N′,N′-tetramethyl-uronium-hexafluoro-phosphate(HBTU) or 2-(6-chloro-1-H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminiumhaxafluorophosphate (HCTU) can be used (e.g., in site). Coupling anddeprotection reactions can be monitored using ninhydrin, trinitrobenzenesulphonic acid or isotin test procedures. Upon completion of synthesis,peptides are cleaved from the resin support with concomitant removal ofside-chain protecting groups, e.g., by treatment with 95%trifluoroacetic acid containing about 5-50% scavenger. Scavengerscommonly used are TIPS (triisopropylsilane), ethanedithiol, phenol,anisole water, and mixtures thereof. The exact choice depends on theconstituent amino acids of the peptide being synthesised. For methioninecontaining peptides one can use, e.g., a mixture of TIPS (e.g., 2-5%)and ethanedithiol (e.g., 2-5%).

Trifluoroacetic acid can subsequently be removed by evaporation invacuo, with subsequent trituration with diethyl ether affording thecrude peptide. Any scavengers present can be removed by a simpleextraction procedure which on lyophilisation of the aqueous phaseaffords the crude peptide free of scavengers.

Reagents for peptide synthesis are generally available, e.g., fromCalbiochem-Novabiochem (UK), or EMD4Biosciences (U.S.).

Purification of the peptides can be effected by any one, or acombination of, techniques such as size exclusion chromatography,ion-exchange chromatography, affinity chromatography, differentialsolubility, and reverse-phase high performance liquid chromatography.Analysis of peptides can be carried out using thin layer chromatography,reverse-phase high performance liquid chromatography, mass spectroscopy(e.g., LC-MS), amino-acid analysis after acid hydrolysis and by fastatom bombardment (FAB) mass spectrometry.

SPOT-synthesis, which allows the positional addressable, chemicalsynthesis of peptides on continuous cellulose membranes can be also used(see, e.g., R. Frank, Tetrahedron (1992) 48, 9217).

When the procoagulant peptide is particularly large, e.g., larger than50 amino acids, or larger than 100 amino acids, the procoagulantcompounds of the present disclosure can be made semirecombinantly (see,e.g., U.S. Pat. No. 7,381,408; Dawson et al. Ann. Rev. Biochem. 69:923-9600 (2000); Mei, B. et. al., Blood 116:270-279 (2010); and U.S.Pat. Appl. Publ. US2006/0115876, each of which is incorporated herein inits entirety). In one embodiment, a clotting factor or procoagulantpolypeptide is produced recombinantly, and then attached to anintermediate compound comprising the cleavable substrate andself-immolative spacer via chemical ligation as described herein inExample 1. Chemical ligation can be performed using established organicchemistry techniques and commercially available reagents.

The procoagulant compounds of the invention can be assembled byconjugating the different moieties disclosed herein (e.g., polypeptides,heterologous moieties, linkers, protease-cleavable substrates, etc.)using orthogonal conjugation strategies know in the art. In someembodiments, such strategies include, e.g., alkyne, azide, N-terminalCys, strained alkyne, ketone, aldehyde, tetrazine-trans-cyclooctene, andcombinations thereof.

In one aspect, the procoagulant compounds of the invention can beproduced by using a cleavable polypeptide comprising a proteasecleavable site, e.g., SUMO. Small Ubiquitin-like Modifier (or SUMO) is amember of the ubiquitin (Ub) and ubiquitin-like (Ubl) family.Post-translational attachment of SUMO to target proteins occurs throughan enzymatic cascade analogous to the ubiquitin conjugation cascade(E1-E2-E3 enzymes), ultimately resulting in formation of an isopeptidebond between the Ub/Ubl C-terminal residue and substrate lysine residue.

SUMO Protease, a highly active cysteinyl protease also known as Ulp, isa recombinant fragment of Ulp1 (Ubl-specific protease 1) fromSaccharomyces cerevisiae. SUMO Protease cleaves in a highly specificmanner, recognizing the tertiary structure of the ubiquitin-like (UBL)protein, SUMO, rather than an amino acid sequence. The protease can beused to cleave SUMO from recombinant fusion proteins. The sequence ofthe SUMO protein comprises:

(SEQ ID NO: 90) SLQDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQAPEDLDMEDNDIIEAHREQIGG

In one embodiment, a cleavable polypeptide useful for the inventioncomprises a light chain of a clotting factor, a truncated heavy chain ofthe clotting factor, and a protease cleavable site, wherein the proteasecleavable site is inserted between the truncated heavy chain of theclotting factor and the light chain of the clotting factor. In anotherembodiment, the cleavable polypeptide further comprises an intracellularprocessing site (also referred herein as proprotein convertaseprocessing site) between the protease cleavable site and the light chainof the clotting factor. In other embodiments, a cleavable polypeptidecomprises a light chain of a clotting factor, a protease cleavable site,a truncated heavy chain of the clotting factor, and a heterologousmoiety, wherein the protease cleavable site is inserted between thelight chain and the truncated heavy chain of the clotting factor, andthe heterologous moiety is linked to the truncated heavy chain of theclotting factor by an optional linker. In still other embodiments, theprotease cleavable site comprises SUMO, which can be cleaved by a SUMOprotease.

In some embodiments, the heterologous moiety comprises a half-lifeextending moiety. Non-limiting examples of the half-life extendingmoiety are disclosed elsewhere herein. In certain embodiments, theclotting factor for the cleavable polypeptide comprises FVII or FX. Insome embodiments, the truncated heavy chain of the clotting factor doesnot comprise one or more amino acids at the N-terminus of the truncatedheavy chain compared to the wild-type heavy chain of the clottingfactor. In certain embodiments, the one or more amino acids deleted fromthe truncated heavy chain of the clotting factor are two amino acids,three amino acids, four amino acids, five amino acids, six amino acids,seven amino acids, eight amino acids, nine amino acids, ten amino acids,eleven amino acids, twelve amino acids, thirteen amino acids, fourteenamino acids, or fifteen amino acids corresponding to the N-terminus ofthe heavy chain that are missing from the truncated heavy chain of theclotting factor. In one example, the clotting factor for the cleavablepolypeptide is Factor VII and the one or more amino acids are IVGGKV(SEQ ID NO: 83). In another example, the clotting factor for thecleavable polypeptide is Factor X and the one or more amino acids areIVGGQE (SEQ ID NO: 85).

In certain embodiments, the invention is directed to a method of makinga cleavable polypeptide comprising transfecting a host cell with apolynucleotide or a vector encoding a cleavable polypeptide under acondition sufficient to express the polypeptide. In some embodiments,the host cell further expresses a proprotein convertase, e.g., PACE orPC5, that can process any intracellular processing sites, e.g., 2X (RKR)(SEQ ID NO: 88) or RRRR (SEQ ID NO: 89).

In some aspects, a cleavable polypeptide comprising a light chain of aclotting factor, a protease cleavable site, and a truncated heavy chainof the clotting factor is cleaved by a protease. After the cleavage, theresulting construct therefore can comprise at least two chains, thefirst chain comprising the light chain of the clotting factor and thesecond chain comprising the truncated heavy chain of the clottingfactor. In some embodiments, the truncated heavy chain of the clottingfactor lacks one or more amino acids at the end of the N-terminuscompared to the wild-type clotting factor, thereby exposing Cysteine atthe N-terminus for chemical ligation. In one embodiment, the amino acidsmissing from the truncated heavy chain are six amino acids, e.g., IVGGKV(SEQ ID NO: 83) for FVII or IVGGQE (SEQ ID NO 85) for FX. In anotherembodiment, the amino acids missing from the heavy chain are 11 aminoacids, e.g., IVGGKVCPKGE (SEQ ID NO: 84) for FVII or IVGGQECKDGE (SEQ IDNO: 86) for FX). In some embodiments, the protease cleavable sitecomprises SUMO.

In one aspect, the invention comprises a method of making a procoagulantcompound comprising combining the cleavable polypeptide with a proteaseunder a condition sufficient to cleave the protease cleavable site. Inanother embodiment, the method further comprises adding a thioesterpeptide to the cleaved polypeptide. The thioester peptide can then befused to the N-terminus of the truncated heavy chain of the clottingfactor, forming activatable clotting factor. In one example, thethioester peptide comprises a protease-cleavable substrate (Zy). Inanother embodiment, the thioester peptide comprises a protease-cleavablesubstrate and a self-immolative spacer (Bx), wherein the self-immolativespancer is inserted between the protease-cleavable substrate and thetruncated heavy chain. In other embodiments, the thioester peptidecomprises a protease-cleavable substrate, a self-immolative spacer, andone or more amino acids (W), wherein the self-immolative spacer isinserted between the protease-cleavable substrate and the one or moreamino acids (W). In one aspect, the one or more amino acids comprise theN-terminus amino acid sequence missing from the truncated heavy chain ofthe clotting factor compared to the wild-type heavy chain. Therefore, incertain embodiments, the thioester peptide comprising aprotease-cleavable substrate, a self-immolative moiety, and one or aminoacids (e.g., six amino acids corresponding to wild-type heavy chain)forms protease-cleavable clotting factor, which comprises theprotease-cleavable substrate, the self-immolative moiety, andfull-length heavy chain. In some embodiments, the one or more aminoacids comprise IVGGKV (SEQ ID NO: 83), wherein the clotting factorcomprises Factor VII. In certain embodiments, the one or more aminoacids comprise IVGGQE (SEQ ID NO: 85), wherein the clotting factorcomprises Factor X. In other embodiments, the one or more amino acidscomprise 11 amino acids, e.g., IVGGKVCPKGE (SEQ ID NO: 84) for FVII orIVGGQECKDGE (SEQ ID NO: 86) for FX). In yet other embodiments, thethioester peptide comprises a formula: Zy-Bx-W, wherein Zy is aprotease-cleavable substrate; Bx is a self-immolative spacer; and W isone or more amino acids that are missing from the truncated heavy chainof the clotting factor. In a particular embodiment, the one or moreamino acids and the truncated heavy chain of the clotting factor, whenfused, result in the complete heavy chain of the clotting factor. Instill other embodiments, the protease-cleavable substrate comprises athrombin cleavage site, e.g., D-Phe-Pip-Arg. In yet other embodiments,the self-immolative spacer comprises PABC.

A variety of methods are available for recombinantly producing aclotting factor or procoagulant peptide for subsequent incorporation ina procoagulant compound of the invention. For recombinant production, apolynucleotide sequence encoding the clotting factor or procoagulantpeptide is inserted into an appropriate expression vehicle, i.e., avector which contains the necessary elements for the transcription andtranslation of the inserted coding sequence, or in the case of an RNAviral vector, the necessary elements for replication and translation.The nucleic acid encoding the clotting factor or procoagulant peptide isinserted into the vector in proper reading frame. The expression vectoris then transfected into a suitable target cell which will express theprocoagulant polypeptide. Transfection techniques known in the artinclude, but are not limited to, calcium phosphate precipitation (Wigleret al. 1978, Cell 14: 725) and electroporation (Neumann et al. 1982,EMBO, J. 1: 841). A variety of host-expression vector systems can beutilized to express the procoagulant polypeptides described herein ineukaryotic cells. In one embodiment, the eukaryotic cell is an animalcell, including mammalian cells (e.g. 293 cells, PerC6, HO, BHK, Cos,HeLa cells).

V. Methods of Treatment

The present invention further provides methods for treating,ameliorating or preventing a bleeding disease or disorder in a subject(e.g., a human subject). An exemplary method comprises administering tothe subject in need thereof a therapeutically effective amount of aprocoagulant compound or a pharmaceutical composition/formulation of thepresent disclosure. In some embodiments, the procoagulant compounds orthe pharmaceutical composition of the invention is administered to thesubject orally.

The procoagulant compounds and pharmaceutical compositions of theinvention can be used prophylactically. As used herein the term“prophylactic treatment” refers to the administration of a moleculeprior to a bleeding episode. In one embodiment, the subject in need of ageneral hemostatic agent is undergoing, or is about to undergo, surgery.The procoagulant compound or pharmaceutical composition of the inventioncan be administered prior to or after surgery as a prophylactic. Theprocoagulant compound or pharmaceutical composition of the invention canbe administered during or after surgery to control an acute bleedingepisode. The surgery can include, but is not limited to, livertransplantation, liver resection, dental procedures, or stem celltransplantation.

The procoagulant compound or pharmaceutical composition of the inventioncan also used for on-demand treatment. The term “on-demand treatment”refers to the administration of a procoagulant compound orpharmaceutical composition of the invention in response to symptoms of ableeding episode or before an activity that can cause bleeding. In oneaspect, the on-demand treatment can be given to a subject when bleedingstarts, such as after an injury, or when bleeding is expected, such asbefore surgery. In another aspect, the on-demand treatment can be givenprior to activities that increase the risk of bleeding, such as contactsports.

Treat, treatment, treating, as used herein refers to, e.g., thereduction in severity of a disease or disorder; the reduction in theduration of a disease course; the amelioration of one or more symptomsassociated with a disease or disorder; the provision of beneficialeffects to a subject with a disease or disorder, without necessarilycuring the disease or disorder, or the prophylaxis of one or moresymptoms associated with a disease or disorder.

In one example according to any of the above embodiments, the bleedingdisease or disorder is caused by a blood coagulation disorder. A bloodcoagulation disorder can also be referred to as a coagulopathy. In aparticular example, the blood coagulation disorder, which can be treatedwith a compound or a pharmaceutical composition of the currentdisclosure, is hemophilia or von Willebrand disease (vWD). In aparticular example, the blood coagulation disorder, which can be treatedwith a compound or a pharmaceutical composition of the presentdisclosure is hemophilia A.

In another example, the type of bleeding associated with the bleedingdisease or disorder is selected from hemarthrosis, muscle bleed, oralbleed, hemorrhage, hemorrhage into muscles, oral hemorrhage, trauma,trauma capitis, gastrointestinal bleeding, intracranial hemorrhage,intra-abdominal hemorrhage, intrathoracic hemorrhage, bone fracture,central nervous system bleeding, bleeding in the retropharyngeal space,bleeding in the retroperitoneal space, and bleeding in the illiopsoassheath.

In another example, the subject suffering from a bleeding disease ordisorder is in need of treatment for surgery, including, e.g., surgicalprophylaxis or pen-operative management. In one example, the surgery isselected from minor surgery and major surgery. Exemplary surgicalprocedures include tooth extraction, tonsillectomy, inguinal herniotomy,synovectomy, craniotomy, osteosynthesis, trauma surgery, intracranialsurgery, intra-abdominal surgery, intrathoracic surgery, jointreplacement surgery (e.g., total knee replacement, hip replacement, andthe like), heart surgery, and caesarean section.

A coagulation disorder can be caused by a deficiency in at least oneblood coagulation factor (e.g., FVIII). The current disclosure providesa method of treating a subject (e.g., a human subject) having adeficiency in at least one blood coagulation factor selected from vonWillebrand Factor (vWF), FV, FVII, FVIIa, FVIII, FIX, FIXa, FX, FXI, andFXa (e.g., for both the prophylaxis and for the treatment of acutebleeds). An exemplary method comprises administering to the subject atherapeutically effective amount of a procoagulant compound or apharmaceutical composition of the present disclosure.

In one example according to any of the above embodiments, the subject isa human subject (i.e., a human patient). In another example according toany of the above embodiments, the subject (e.g, human patient) isconcomitantly treated with at least one additional active agent, e.g., adrug approved for the treatment of coagulation disorders. In oneexample, the additional active agent is administered to the subject atthe same time that the procoagulant compound or pharmaceuticalcomposition of the present disclosure is administered to the subject.For example, the at least one additional active agent is contained in apharmaceutical composition that also contains the compound of thepresent disclosure. In another example, the additional active agent isadministered to the subject at a different time but within the treatmentperiod for the compound of the present disclosure. For example, theadditional active agent is administered alternatingly with theprocoagulant compound or pharmaceutical composition of the presentdisclosure.

For oral and parenteral administration to patients, including humanpatients, the daily dosage level of the procoagulant compound of thecurrent disclosure will usually be from 2 to 2000 mg per adult (i.e.from about 0.03 to 30 mg/kg), administered in single or divided doses.

A unit dosage form (for example tablet or capsule) can contain from 2 mgto 2000 mg of procoagulant compound. The unit dosage form can beadministered once, twice or more times per day as appropriate. Thephysician in any event will determine the actual dosage which will bemost suitable for any individual patient and it will vary with the age,weight and response of the particular patient. The above dosages areexemplary of the average case. There can, of course, be individualinstances where higher or lower dosage ranges are merited and such arewithin the scope of this invention.

VI. Other Methods

The invention further provides a method of increasing the efficacy ofthe cleavage of a protease-cleavage substrate (e.g., athrombin-cleavable substrate) operably linked to a procoagulantpolypeptide (e.g., a synthetic procoagulant peptide or clotting factor)comprising conjugating a self-immolative linker (e.g., PABC) to theprocoagulant polypeptide, wherein the self-immolative linker isinterposed between the protease-cleavage substrate and the procoagulantpolypeptide.

In some embodiments, the efficacy of cleavage is increased by at leastabout 10%, by at least about 20%, by at least about 30%, by at leastabout 40%, by at least about 50%, by at least about 60%, by at least70%, by at least about 80%, by at least 90% or by at least about 100%when compared to a reference procoagulant compound with the samesequence but without a self-immolative linker. In some embodiments, theefficacy of cleavage is increased by at least 100% when compared to areference procoagulant compound with the same sequence but without aself-immolative linker.

In some embodiments, the efficacy of cleavage is at least about 2-fold,at least about 3-fold, at least about 4-fold, at least about 5-fold, atleast about 6-fold, at least about 7-fold, at least about 8-fold, atleast about 9-fold, or at least about 10-fold when compared to areference procoagulant compound with the same sequence but without aself-immolative linker. In some embodiments, the efficacy of cleavage isat least about 20-fold, at least about 30-fold, at least about 40-fold,at least about 50-fold, at least about 60-fold, at least about 70-fold,at least about 80-fold, at least about 90-fold, or at least about100-fold when compared to a reference procoagulant compound with thesame sequence but without a self-immolative linker.

In some embodiments, wherein the procoagulant compound is cleaved by aprotease specific for the protease-cleavable substrate moiety at least10% faster when compared to a reference procoagulant compound with thesame sequence but without a self-immolative linker. In some embodiments,cleavage is at least about 10%, at least about 20%, at least about 30%,at least about 40%, at least about 50%, at least about 60%, at least70%, at least about 80%, at least 90% or at least about 100% faster whencompared to a reference procoagulant compound with the same sequence butwithout a self-immolative linker. In some embodiments, the cleavage isfaster by at least 100% when compared to a reference procoagulantcompound with the same sequence but without a self-immolative linker. Insome embodiments, the cleavage is at least about 2-fold, at least about3-fold, at least about 4-fold, at least about 5-fold, at least about6-fold, at least about 7-fold, at least about 8-fold, at least about9-fold, or at least about 10-fold faster when compared to a referenceprocoagulant compound with the same sequence but without aself-immolative linker.

Also provided in the present disclosure is a method of activating aprocoagulant peptide, comprising contacting a procoagulant compound ofthe invention a protease specific for the protease-cleavable substratemoiety in said procoagulant compound, wherein the activated procoagulantpeptide is released upon proteolytic cleavage of the protease-cleavablesubstrate moiety.

The disclosure also provides a method of activating a clotting factorcomprising contacting a procoagulant compound of the invention with aprotease specific for the protease-cleavable substrate moiety in saidprocoagulant compound, wherein the activated clotting factor is releasedupon proteolytic cleavage of the protease-cleavable substrate moiety. Insome embodiments, more than one clotting factor (e.g., an activatedclotting factor), clotting factor fragment (e.g., a heavy chain or alight chain), procoagulant peptide (e.g., a synthetic procoagulantpeptide) or combinations thereof can be released upon proteolyticcleavage of the procoagulant compound of the invention.

The disclosure also provides a method of releasing a procoagulantpeptide from a heterologous moiety comprising contacting a procoagulantcompound of the invention with a protease specific for theprotease-cleavable substrate in said procoagulant compound, wherein theactivated procoagulant polypeptide is released upon proteolytic cleavageof the protease-cleavable substrate.

The disclosure also provides a method of releasing a clotting factorfrom a heterologous moiety comprising contacting a procoagulant compoundof the invention with a protease specific for the protease-cleavablesubstrate in said procoagulant compound, wherein the activated clottingfactor is released upon proteolytic cleavage of the protease-cleavablesubstrate. In some embodiments, more than one clotting factor (e.g., anactivated clotting factor), clotting factor fragment (e.g., a heavychain or a light chain), procoagulant peptide (e.g., a syntheticprocoagulant peptide) or combinations thereof can be released uponproteolytic cleavage of the procoagulant compound of the invention fromone or more heterologous moieties (e.g., PEG). In some embodiments, therelease of the one or more than one clotting factor (e.g., an activatedclotting factor), clotting factor fragment (e.g., a heavy chain or alight chain), procoagulant peptide (e.g., a synthetic procoagulantpeptide) or combinations thereof factor and the heterologous moietytakes place simultaneously. In some embodiments, the release of therelease of the one or more than one clotting factor (e.g., an activatedclotting factor), clotting factor fragment (e.g., a heavy chain or alight chain), procoagulant peptide (e.g., a synthetic procoagulantpeptide) or combinations thereof factor and the release of theheterologous moiety takes place sequentially.

Also provided in the disclosure is a method of releasing at least oneheterologous moiety from a procoagulant compound of the invention, prioror concurrently with the release of one or more clotting factors,clotting factor fragments (e.g., a heavy chain or a light chain),procoagulant peptides (e.g., a synthetic procoagulant peptide) orcombinations thereof by treatment with one or more proteases, comprisingcontacting a procoagulant compound of the invention with one or moreproteases specific for the one or more than one protease-cleavablesubstrate in said procoagulant compound. In some embodiments, therelease of one, two, three or more than three heterologous moietiestakes place prior to the proteolytic release of clotting factors,clotting factor fragments (e.g., a heavy chain or a light chain),procoagulant peptides (e.g., a synthetic procoagulant peptide) orcombinations thereof. In other embodiments, the release of one, two,three or more than three heterologous moieties takes place concurrentlywith the proteolytic release of clotting factors, clotting factorfragments (e.g., a heavy chain or a light chain), procoagulant peptides(e.g., a synthetic procoagulant peptide) or combinations thereof. Insome embodiments, the release of two, three or more than threeheterologous moieties takes place simultaneously. In some embodiments,the release of two, three or more than three heterologous moieties takesplace sequentially.

Having now described the present invention in detail, the same will bemore clearly understood by reference to the following examples, whichare included herewith for purposes of illustration only and are notintended to be limiting of the invention. All patents, patentapplication, patent application publications, and other publicationsreferred to herein are expressly incorporated by reference in theirentireties.

EXAMPLES Materials and Methods

The materials and methods for peptide synthesis, purification, andcharacterization described below are used in the Examples below unlessotherwise stated.

1. Solid Phase Peptide Synthesis

Synthetic procoagulant peptides of the present disclosure weresynthesized by solid phase peptide synthesis using9-fluorenylmethoxycarbonyl/tertiary-butyl (Fmoc/tBu) chemistry. Heatingwas accomplished using a microwave oven or other means. In most cases,the peptides were synthesized in 0.1 mmol scale using NovaPEG Rink Amideresin (Novabiochem) or NovaPEG TGT resin (Novabiochem) in a 35 mLreaction vessel. Standard methods for resin load, amino acid coupling,Fmoc deprotection and washing steps were performed on a CEM Libertypeptide synthesizer (CEM Corp.), whereas the trifluoroacetic acid (TFA)cleavage of the peptide was performed manually.

Briefly, 5 equivalent of Fmoc protected amino acids dissolved inN,N-dimethylformamide (DMF) were linked subsequently to the resin in thepresence of 5 equivalents of2(6-chloro-1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate (HCTU) and 10 equivalents of diisopropylethylamine (DIPEA).The microwave method used for the coupling step was single coupling at75° C. (20 W for 300 seconds), except for cysteine and histidine, whichwere coupled at 50° C. (0 W for 120 sec, 20 W for 240 seconds). Argininewas double coupled at 75° C. (0 W for 1500 sec, 20 W for 300 seconds).The Fmoc deprotection was performed with 5% piperazine, 0.1M1-hydroxybenzotriazole (HOBt) in DMF at 75° C. (45 W for 30 seconds, 45W for 180 seconds). Most amino acids and coupling reagents werepurchased from Novabiochem® EMD (EMD Millipore Chemicals).

Following the automated peptide synthesis, the peptides were cleavedfrom the resin with 95% TFA and 5% triisopropylsilane (TIPS) for 2 hoursor 30% hexafluoroisopropanol (HFIP) in DCM. Next, the peptides werefiltered into round bottom reaction flasks. The solvents were removed invacuo, and the concentrates containing the peptides were precipitatedand further triturated with ice cold diethyl ether (Et₂O). The identityof the synthesized peptides was confirmed by mass spectral analysis.

2. Peptide Purification

The synthesized peptides were purified by preparative reverse phase highperformance liquid chromatography (RP-HPLC) using Waters 600 controllerand pump system equipped with a Waters 2489 UV/Visible detector and aWater Fraction Collector III (Waters Corp.). The purifications weretypically performed on a Phenomenex Jupiter C18 10 micron 250×21.20 mmRP-HPLC column (Phenomenex, Inc.) with a flow rate of 20 mL/min. Theacetonitrile/water (0.1% TFA) gradient was modified for each specificpeptide based on hydrophobicity. The peptides were detected at twowavelengths, 228 nm and 280 nm, and the fractions were further analyzedby liquid chromatography mass spectrometry (LC-MS). Fractions containingpeptide of adequate purity were pooled, flash frozen and lyophilized.

3. Peptide Characterization

The peptides were characterized by LC-MS (Agilent LC-MS TOF 6220 with1200 series pump, auto handler and UV detection system). The LCseparation was performed on a Phenomenex Jupiter C18 5 micron 250×2.00mm column using a mobile phase of A (water+0.08% formic acid+0.02%trifluoroacetic acid) and B (acetonitrile+0.08% formic acid+0.02%trifluoroacetic acid). The general LC method had a gradient from 0-70% Bover 12 min. Mass determination was achieved by electrospray ionizationin positive mode. The purity of the peptides was determined by measuringthe absorbance of UV light at 228 nm over the chromatogram.

Example 1 Thrombin-Activatable Procoagulant Compounds with PABCSelf-Immolative Linker

Seven different peptides, designated Compound 1 to 7, were used in theexperiments disclosed herein (TABLE 1). The sequenceIle-Val-Gly-Gly-Gln-Glu in Compounds 1 to 6 corresponds to the sixN-terminal amino acid residues of the heavy chain of the FXa clottingfactor. These compounds reproduce the coupling of a thrombin cleavablesubstrate and a self-immolative spacer to the N-terminus of a clottingfactor or a fragment thereof, in this specific example, FX. Compound 7corresponds to a synthetic procoagulant peptide fused to PABC and to athrombin-cleavable substrate, and further including a linker and ascaffolding amino acid heterologous moiety (Cys) for attachment ofhalf-life extending moieties such as PEG.

TABLE 1 Com- pound Structure 1

2 (D-Phe)-Pip-Arg-Ile-Val-Gly-Gly-Gln-Glu-NH₂ 3Ala-Leu-Arg-Pro-Arg-Ile-Val-Gly-Gly-Gln-Glu-NH₂ 4

5

6 Ala-Leu-Val-Pro-Arg-Ile-Val-Gly-Gly-Gln-Glu-NH₂ 7

Leu-Ala-Ser-Tyr-Cys-Trp-Leu-Phe-Trp-Thr-Gly-Ile-Ala-NH₂

Pip is pipecolic acid. (D-Phe) is D-Phenyl alanine. The sequences of thethrombin substrate are underlined. The location of the PABCself-immolative linker is indicated by a box.1. Synthesis of PABC Peptides (Compound 1, 4, 5, and 7)

The synthesis process for Compound 7 is shown in FIG. 5 and FIG. 6, andexplained in detailed below. The synthesis of Compounds 1, 4, and 5 wasoutsourced, and followed a similar synthesis procedure. Compounds 2, 3,6 were synthesized as described in the Materials and Methods section,supra.

The first two steps in the synthesis of Compound 7 after cleavage fromthe resin are shown in FIG. 3:

Compound A Synthesis

Fmoc-Cys(Acm)-Gly-Gly-Gly-Gly-Dphe-Pip-Arg(Pbf)

The chemically synthesized and fully protected Compound A peptide(Fmoc-Cys(Acm)-Gly-Gly-Gly-Gly-Dphe-Pip-Arg(Pbf)) was cleaved from theNovaPEG TGT resin by 30% HFIP/DCM and filtered into a round bottomreaction flask. The solvents were removed in vacuo, and the concentratecontaining the peptide was precipitated and further triturated with icecold diethyl ether (Et₂O). This material was directly used withoutfurther purification. ESI-MS m/z: 1309.51 (MH)⁺.

Compound B Synthesis

Fmoc-Cys(Acm)-Gly-Gly-Gly-Gly-Dphe-Pip-Arg(Pbf)-PABOH (p-Amino BenzylAlcohol)

A stirred solution of Fmoc-Cys(Acm)-Gly-Gly-Gly-Gly-Dphe-Pip-Arg(Pbf)(Compound A) (268 mg, 0.2 mmol) and p-amino benzyl alcohol (28 mg, 1.1equivalents) in THF (2 mL) at room temperature was treated with EEDQ(55.6 mg, 1.1 equivalents). After 16 hours, the mixture was evaporatedto dryness, and the residue was triturated with ether. The resultingwhite solid product, comprising Compound B(Fmoc-Cys(Acm)-Gly-Gly-Gly-Gly-Dphe-Pip-Arg(Pbf)-PABOH (p-amino benzylalcohol)) was collected by centrifugation and dried in vacuo (200 mg,70%). ESI-MS m/z: 1414.61 (MH)⁺.

Compound C Synthesis

Fmoc-Cys(Acm)-Gly-Gly-Gly-Gly-Dphe-Pip-Arg(Pbf)-PABC-PNP

A stirred solution ofFmoc-Cys(Acm)-Gly-Gly-Gly-Gly-Dphe-Pip-Arg(Pbf)-PABOH (Compound B) (180mg, 0.127 mmol) in dry THF (4 mL) and DCM (4 mL) at room temperature wastreated with PNP chloroformate (38.5 mg, 1.5 equivalents) and drypyridine (15 mg, 1.5 equivalents). After 16 hours, the mixture wasconcentrated to 1 mL, and the product was precipitated and trituratedwith cold ether. The resulting white solid product, comprising CompoundC (Fmoc-Cys(Acm)-Gly-Gly-Gly-Gly-Dphe-Pip-Arg(Pbf)-PABC-PNP) wascollected by centrifugation and dried in vacuo (150 mg, 75%). ESI-MSm/z: 1579.61 (MH)⁺.

The remaining steps in the synthesis of Compound 7, comprising theconjugation of the protease-cleavable substrate/self-immolative spacerto the synthetic procoagulant peptide are depicted in FIG. 4.

Compound D Synthesis

rRAPGK(Alloc)LTCLASYCWLFWTGIA-NH₂ (disulfide)

The linear peptide was synthesized on NovaPEG Rink Amide resin (0.2mmol) as described in the general method. The Cys-Cys disulfidic bondwas formed by stirring the crude peptide in 50% DMSO/H₂O overnight at37° C. 35 mg of peptide was obtained after purification by preparativeHPLC. ESI-MS m/z: 1298.17 (MH₂)²⁺, 865.78 (MH₃)³⁺.

Compound E Synthesis

Fmoc-Cys(Acm)-Gly-Gly-Gly-Gly-Dphe-Pip-Arg(Pbf)-PABC-rRAPGK(Alloc)LTCLASYCWLFWTGIA-NH₂ (disulfide)

Fmoc-Cys(Acm)-Gly-Gly-Gly-Gly-Dphe-Pip-Arg(Pbf)-PABC-PNP (Compound C)(12.5 mg, 0.008 mmol) and rRAPGK(Alloc)LTCLASYCWLFWTGIA (Compound D) (30mg, 0.011 mmol) in DMF (1 mL) at room temperature were treated with DIEA(6.5 μL, 5 equivalents). The mixture was allowed to stand in the darkovernight. The crude product was precipitated, and triturated with coldether. The resulting crude product was collect by centrifugation, driedin vacuo, and used for next step without further purification. ESI-MSm/z: 2018.32 (MH₂)²⁺, 1345.86 (MH₃)³⁺.

Compound 7 Synthesis

Step 1

Fmoc-Cys(Acm)-Gly-Gly-Gly-Gly-Dphe-Pip-Arg-PABC-rRAPGK(Alloc)LTCLASYCWLFWTGIA-NH₂ (disulfide)

Pbf deprotection ofFmoc-Cys(Acm)-Gly-Gly-Gly-Gly-Dphe-Pip-Arg(Pbf)-PABC-rRAPGK(Alloc)LTCLASYCWLFWTGIA from the previous step was carried out in 1 mL of solventmixture (72% TFA, 5% DMF, 5% H₂O, 18% DCM) for 75 minutes. Since thePABC linker was unstable under this condition, aliquots were taken atvarious time points to monitor progress of the reaction. At 75 minutes,cold ether (50 mL) was added to stop the reaction. The resulting solidwas purified by preparative HPLC, to give a white powder (8 mg, 25% for2 steps). ESI-MS m/z: 1261.83 (MH₃)³⁺.

Step 2

Fmoc-Cys(Acm)-Gly-Gly-Gly-Gly-Dphe-Pip-Arg-PABC-rRAPGKLTCLASYCWLFWTGIA-NH₂ (disulfide)

Fmoc-Cys(Acm)-Gly-Gly-Gly-Gly-Dphe-Pip-Arg-PABC-rRAPGK(Alloc)LTCLASYCWLFWTGIA (8mg, 0.002 mmol) in MeOH/Dioxane (1:1, 180 μL) under N₂ at roomtemperature was treated with Pd(PPh₃)₄ (0.0002 mmol, 0.1 equivalents, 20μL of a THF solution of Pd(PPh₃)₄ (23 mg/mL), followed by PhSiH₃ (0.01mmol, 5 equivalents). After 20 minutes, the crude mixture wasprecipitated and triturated with cold ether. The resulting crude productwas used for the next step without purification. ESI-MS m/z: 1233.82(MH₃)³⁺.

Step 3

Cys(Acm)-Gly-Gly-Gly-Gly-Dphe-Pip-Arg-PABC-rRAPGKLTCLASYCWLFWTGIA-NH₂(disulfide) (Compound 7)

Fmoc deprotection ofFmoc-Cys(Acm)-Gly-Gly-Gly-Gly-Dphe-Pip-Arg-PABC-rRAPGKLTCLASYCWLFWTGIAfrom the previous step was carried out in DMSO (200 μL) with Et₂NH (50μL, excess). After 20 minutes, the reaction was complete and the mixturewas purified by preparative HPLC, to give a white powder SYN 4018 (0.83mg, 12% over 2 steps). ESI-MS m/z: 1159.80 (MH₃)³⁺, 870.09 (MH₄)⁴⁺.

2. Thrombin Cleavage of Compound 7

FIG. 5 depicts the cleavage of Compound 7 by thrombin. Upon cleavagewith 1.4 nM thrombin in PBS, the clean synthetic procoagulant peptide isreleased, as well as the portion of the molecule comprising the thrombinsubstrate, the GGGG linker, and the N-terminal cysteine. To conduct thereaction, 21 μL of peptide (0.24 mM) in water was added to 476.5 μL PBS.The mixture was incubated at 37° C. for 30 min, followed by 2.5 μL ofthrombin (278 nM, 10 μg/mL), giving the following approximate initialconcentrations: thrombin=1.4 nM, peptide=10 μM. The mixture wasincubated at 37° C. Aliquots (60 μL) at various time points werequenched with 1 μL of hirudin (10 μM) and injected into the HPLC (C-18column, CH₃CN/H₂O, 0 to 70% over 12 minutes, 60° C. 0.5 mL/min,

=280 nm). The kinetics of the cleavage of Compound 7 is shown in FIG. 6.Compound 7 was cleaved rapidly by 1.4 nm thrombin. Approximately 90% ofCompound 7 was cleaved after 30 minutes.

Compound 7 was also cleaved completely during the course of a TGA assay(FIG. 7). Plasma at the end of TGA assay for Compound 7 was transferredinto cold CH₃CN (1 mL) and centrifuged at 13 k rpm for 10 min.Supernatant (1.1 mL) was transferred to a new vial and dried byspeedvac. The resulting solid was reconstituted with 30 μL of H₂O andinjected into the HPLC for analysis. FIG. 7 shows peaks corresponding tothe procoagulant peptide in Compound 7, indicating that Compound 7 wascompletely cleaved in the course of the TGA assay.

3. Thrombin Cleavage of Compounds 1, 2, and 3

FIG. 8 depicts the cleavage of Compounds 1, 2 and 3 by 14 nM thrombin.These compounds, as discussed above, comprise the six N-terminal aminoacid residues of the heavy chain of the FXa clotting factor, andfunction as a model to show the applicability of the procoagulantcompound design disclosed herein to clotting factors.

In this specific example, 50 μL of peptide (1 mM) in water was added to900 μL PBS, followed by 50 μL of thrombin (278 nM, 10 μg/mL), giving thefollowing approximate initial concentrations: thrombin=14 nM, peptide=50μM. The mixture was incubated at room temperature. Aliquots (95 μL) atvarious time points were quenched with 5 μL of hirudin (2 μM) andinjected into the HPLC (C-18 column, CH₃CN/H₂O, 0 to 70% over 12minutes, 60° C. 0.5 mL/min,

=280 nm). The decreases of peptide peak areas were used to calculateyield.

Compared to Compounds 2 and 3, the construct incorporating thethrombin-cleavable synthetic substrate D-Phe-Pip-Arg (SEQ ID NO: 21) andthe self-immolative spacer PABC (Compound 1) was a better substrate forthrombin. The incorporation of PABC to Compound 1 led to at least10-fold increase in cleavage rate compared to that of Compound 2.

4. Thrombin Cleavage of Compounds 1, 4, 5 and 6

FIG. 9 depicts the cleavage of Compounds 1, 4, 5 and 6 by 1.4 nMthrombin. Compounds 1, 4 and 5 incorporate PABC and differentthrombin-cleavable substrates.

50 μL of peptide (1 mM) in water was added to 900 μL PBS. The mixturewas incubated at 37° C. for 30 min, followed by 50 μL of thrombin (27.8nM, 1 μg/mL), giving the following approximate initial concentrations:thrombin=1.4 nM, peptide=50 μM. The mixture was incubated at 37° C.Aliquots (95 μL) at various time points were quenched with 5 μL ofhirudin (2 μM) and injected into the HPLC (C-18 column, CH₃CN/H₂O, 0 to70% over 12 minutes, 60° C. 0.5 mL/min,

=280 nm). The decreases of peptide peak areas were used to calculateyield.

Compound 1 was a better substrate for thrombin than Compounds 4 and 5.At 1.4 nM, a physiological relevant concentration of thrombin, 30% ofCompound 1 was quickly cleaved and released. In contrast,thrombin-mediated release of peptide IVGGQE (SEQ ID NO: 85) fromCompound 6 without PABC linker was not observed.

Example 2 Thrombin-Activatable FX with PABC Self-Immolative Linker

Peptide synthesis method equivalents to those described above, standardrecombinant protein production methods, and standard chemicalconjugation techniques are used to generate the procoagulant compounddescribed in this example.

Factor X consists of two polypeptide chains linked by a disulfide bridge(Cys172-Cys342): the 139 amino acid light chain in composed of the Gladomain and the two EGFs; the 306 amino acid heavy chain is composed ofthe activation peptide joined to the catalytic domain. The activation offactor X requires proteolytic cleavage between the activation peptideand the catalytic domain. The tensase complex and the FVIIa-TF complexperform this cleavage between the Arg234 and Ile235 residues (FIG. 10).As in all serine proteases, the N-terminal residues of the catalyticchain of activated factor X are involved in the enzymatic activity. Thegenerated N-terminal Ile235 in particular plays a fundamental role inthe catalytic mechanism of the enzyme.

PCT Publ. No. WO 2004/005347 proposed replacement of the native site foractivation by the tenase complex with a site for cleavage by thrombin.However, since the efficiency of cleavage is conditioned by the natureof the amino acids framing the cleavage site, these thrombin-activatableFX (TA-FX) analogs suffered from slow activation and undesired efficacy.It is therefore desirable to have other TA-FX analogs which wouldexhibit faster cleavage kinetics.

The present disclosure provides TA-FX analogs comprising a syntheticthrombin substrate and a self-immolative spacer (e.g., PABC) linked toFXa (FIG. 11). After proteolytic cleavage of the thrombin substrate(D-PhePipArg) and 1,6 spontaneous fragmentation, the natural sequence ofFXa is released (FIG. 10). The TA-FX is generated semi-syntheticallyusing native chemical ligation chemistry. This process involves thereaction of a recombinantly produced FX fragment containing an Nterminal cysteine residue 241 on the catalytic domain (CysFX) with asynthetically produced thioester peptide to generate a native amide bondat the linkage site. To generate the CysFX protein, the sequencecomprising the six N-terminal amino acid residues of the heavy chainIVGGQE (SEQ ID NO: 85) is truncated from the FX; and the native site foractivation by tensase complex is replaced with a cleavage site, e.g.,for cleavage by PC5.

Example 3 Thrombin-Activatable FVII with PABC Self-Immolative Linker

Peptide synthesis method equivalent to those described above, standardrecombinant protein production methods, and standard chemicalconjugation techniques are used to generate the procoagulant compoundsdescribed in this example.

The present disclosure provides thrombin-activatable FVII (TA-FVII)analogs comprising a synthetic thrombin substrate and a self-immolativespacer (e.g., PABC) linked to FVIIa (FIG. 13). After proteolyticcleavage of the thrombin substrate (D-PhePipArg) and 1,6 spontaneousfragmentation, the natural sequence of FVIIa is released (FIG. 10). TheTA-FVII is generated semi-synthetically using native chemical ligationchemistry. This process involves the reaction of a recombinantlyproduced FVII fragment containing an N terminal cysteine residue 159 onthe catalytic domain (CysFVII) with a synthetically produced thioesterpeptide to generate a native amide bond at the linkage site. To generatethe CysFVII protein, the sequence comprising the six N-terminal aminoacid residues of the heavy chain IVGGKV (SEQ ID NO: 83) is truncatedfrom the FVII; and the native site for activation by FXa is replacedwith a cleavage site, e.g., for cleavage by PC5.

Example 4 Thrombin Activatable FVII-186 with SUMO Cleavage Site

For cloning of FVII-186, synthesis of the DNA sequence comprisingnucleotides from the HindIII site to the EcoRI site of FVII-186 (Table2) was outsourced. The DNA was subcloned into the HindIII/EcoRI sites ofpcDNA.

To transiently express FVII-186, HEK-293-F cells were grown insuspension in FREESTYLE® media (Invitrogen) supplemented with vitamin K3(Sigma Aldrich, St. Louis, Mo.) to 2 μg/liter (growth media) assuspension cells at 37° C./10% CO₂. Cells were subcultured every threeto four days by seeding at cell density of 5×10⁵ cells/ml. Twenty-fourhours prior to transfection, cells were seeded at a density of 7×10⁵cells/ml in growth media. On the day of transfection, a transfectionsolution was made with a volume equal to 5% of the total volume of thecell culture to be transfected. In the transfection solution, DNA wasadded (final concentration 20 mg/L) to a freshly made solution of PEI(60 mg/L) in growth media. The solution was swirled for 30 seconds andincubated for five minutes at room temperature before adding directly tothe cell culture. Four hours later a volume equal to the cell culturevolume of OPTICHO™ (Invitrogen) supplemented with vitamin K3 and 200 mML-glutamine was added to the cells. The cell culture was allowed to growas shown above and daily media samples were taken to assess proteinexpression. On the day of harvest, the cells were spun down, and themedia filtered in preparation for protein purification or proteinanalysis by protein A pulldown. For expression of FVII-186, a plasmidencoding FVII-186 was contransfected with a plasmid encoding theproprotein convertase PACE to ensure intracellular processing andcleavage of the proprotein convertase cleavage sites (2X(RKR) SEQ ID NO:88) in the linker connecting the FVII light chain to SUMO (FIG. 14).

To purify FVII-186, conditioned medium was loaded onto a 25-mL column ofQ SEPHAROSE® Fast Flow (GE HealthCare Life Sciences) after adjustment ofpH to 7.4 with 2.0 M Tris, pH 8.0. column was washed with 10 mM MES, 50mM NaCl, pH 6.5. The protein was eluted with 10 mM MES, 100 mM NaCl, 20mM CaCl₂, pH 6.5. The fractions containing FVII-186 were pooled andloaded onto a 25-mL column of rhFcRn-sepharose after adjustment of pH to6.2 with 0.5 M MES, pH 5.5. After washing with 50 mM MES, 100 mM NaCl,pH 6.2, the bound material was eluted with 10 mM Tris, 250 mM NaCl, pH8.0 and analyzed with SDS-PAGE.

FVII-186 was cleaved by a SUMO protease as follows. FVII-186 (0.83mg/mL, 10 μL) was incubated with 10 μL of 100 mM HEPES, 20 mM CaCl₂,0.004% Tween 80 containing 0.4 mM oxidized Glutathione (GSSG), 20 mMGlutathione (GSH), 0.2 U/μL SUMO protease (Invitrogen Cat. No.12588-018) for 48 hours at room temperature. Reducing SDS-PAGE (FIG. 15,lane 3) showed almost complete conversion of FVII-186 to the desiredFVIIHC.

For SUMO protease cleavage of FVII-186 and native chemical ligation witha thioester peptide, FVII-186 (0.83 mg/mL, 10 μL) was incubated with 10μL of 100 mM HEPES, 20 mM CaCl₂, 0.004% Tween 80 containing 0.4 mMSYN470 as a positive control peptide, 0.4 mM GSSG, 20 mM GSH, 0.2 U/μLSUMO protease (Invitrogen Cat. No. 12588-018) for 48 hours at roomtemperature. Reducing SDS-PAGE (FIG. 15, lane 4) showed completedisappearance of the FVIIHC band and a single new band as the conjugateof the positive peptide control and the FVIIHC.

In order to synthesize Thrombin Activatable FVII-186 (TA-FVII-186),FVII-186 (0.83 mg/mL, 200 μL) was incubated with 200 μL of 100 mM HEPES,20 mM CaCl₂, 0.004% Tween 80 containing 0.4 mM FVII-PABC peptide (i.e.,Biotin-Pra-GGGG-D-Phe-Pip-Arg-PABC-IVGGKV-COSBn) (SEQ ID NO: 79), 0.4 mMGSSG, 20 mM GSH, 0.2 U/μL SUMO protease (Invitrogen Cat. No. 12588-018)for 48 hours at room temperature and analyzed by reducing SDS-PAGE (FIG.15, lane 5). Reaction mixture was placed in a 0.5 mL dialysis cassettewith 10 k MWCO and dialyzed against 1 L of 10 mM Tris, 250 mM NaCl, pH8.0 containing 0.4 mM GSSG, 2 mM GSH for 24 hours at 4° C. The conjugatewas further purified by rhFcRn-sepharose column as described.

FVIIa Chromogenic assay was performed after Thrombin cleavage andactivation of TA-FVII-186 (FIG. 16). This assay measures the FXactivation activity by measuring the ability of FVIIa to activate FX, asdetermined by measuring levels of a chromogenic substrate that iscleaved by activated FX (FXa). TA-FVII-186 (200 nM) was activated withThrombin (140 nM) for 20 minutes at 37° C. Hirudin was added to quenchThrombin. sTF-PL mixture (A STACLOT® FVII-rTF kit), FX, and PEFACHROME®FXa substrate were added and reaction was monitored by measuringabsorbance at 405 nm. FVII-186 missing the six N-terminal amino acidswas not active in the presence of thrombin. Only TA-FVII-186 with athrombin cleavage site connected to the complete heavy chain FVII (whichincludes FVIIa-PABC peptide) showed activity after thrombin cleavage.The resulted activity demonstrated that the FVIIa-PABC peptide wassuccessfully conjugated to the N-terminal cysteine residue of thetruncated heavy chain of FVIIa, the crucial N-terminal isoleucineresidue was generated upon cleavage by thrombin, and the formed proteinhad the essential structure for activity.

Example 5 Thrombin Activatable FX-011 with PACE Cleavage Site

For cloning of FX-011, synthesis of the DNA sequence comprisingnucleotides from the HindIII site to the NotI site of FX-011 (Table 4)was outsourced. The DNA was subcloned into the HindIII/NotI sites ofpcDNA.

For transient expression of FX-011, HEK-293-F cells were transfectedessentially as described above to obtain expression of FX-011. A plasmidencoding FX-011 was cotransfected with a plasmid encoding the proproteinconvertase PACE (20%) to ensure intracellular processing and cleavage ofthe proprotein convertase cleavage sites in the linkers and removal oflinkers (FIG. 17) In order to analyze the protein from transienttransfections, conditioned media were subjected to protein Aimmunoprecipitation. Briefly, cell culture supernatant was mixed withapproximately 50 μl of protein A-Sepharose 50% slurry and incubated at4° C. with rocking for 1 hour, then centrifuged to pellet the protein Abeads. Beads were washed twice by resuspending in 1 ml of PBS, spinningand aspirating. The beads were resuspended with SDS-PAGE buffer underreducing or nonreducing conditions, heated for 5 minutes at 95° C., spundown, loaded on SDS-PAGE gels, and run according to standard protocols.Under non-reducing conditions, 1 band with the expected molecular weightfor the FX-011 was observed (FIG. 17, lane 3). Under reducingconditions, 3 major bands were observed representing the incompletelyprocessed activation peptide-heavy chain FX-Fc subunit, the desiredheavy chain FX-Fc subunit, and the Fc subunit (FIG. 17, lane 2).Proteins were transferred onto a cellulose membrane and the bandcorresponding to heavy chain FX-Fc subunit was collected and analyzed.N-terminal sequencing confirmed the existing N-terminal cysteine (Cys)residue as expected after cleavage by PACE.

For purification of FX-011, conditioned medium (200 mL) was concentratedto 10 mL by 15 mL centrifugal filter units 30,000 MWCO (catalog #UFC903008). After adjustment of pH to 6.2 with 0.5 M MES, pH 5.5, theconcentrated medium was loaded onto a 0.5 mL rhFcRn-sepharose resin bedequilibrated with 50 mM MES, 100 mM NaCl, pH 6.2 buffer. After washingwith 50 mM MES, 100 mM NaCl, pH 6.2, the bound material was eluted with10 mM Tris, 250 mM NaCl, pH 8.0. Before conjugation, FX-011 wastransferred to a 20 mM HEPES, 500 mM NaCl, 5 mM CaCl₂, pH 7.4 buffer bydialysis.

For semisynthesis of Thrombin Activatable FX-011 (TA-FX-011) by nativechemical ligation with a thioester peptide, FX-011 (0.5 mg/mL) wasincubated with 0.5 mM FX-PABC peptide (i.e.,GG-D-Phe-Pip-Arg-PABC-IVGGQE-COSBn) (SEQ ID No. ______) and 20 mM sodium2-sulfanylethanesulfonate (MESNA) in 20 mM HEPES, 500 mM NaCl, 5 mMCaCl₂, pH 7.4 buffer for 16 hours at room temperature. Reaction wasanalyzed by SDS-PAGE gel (FIG. 18, lane 3). Excess peptides and MESNAwere removed by gel filtration. The pooled fractions containingTA-FX-011 were placed in a 0.5 mL dialysis cassette with 10 k MWCO anddialyzed against 1 L of 20 mM HEPES, 500 mM NaCl, 5 mM CaCl₂, pH 7.4 for24 hours at 4° C.

FXa chromogenic assay was performed after Thrombin cleavage of TA-FX-011(FIG. 19). TA-FX-011 (200 nM) was activated with Thrombin (140 nM) for20 minutes at 37° C. Hirudin was added to quench Thrombin. FXa substratewas added and reaction was monitored by measuring absorbance at 405 nm.FX-011 missing the six N-terminal amino acids was not active in thepresence of thrombin. Only TA-FX-011 with a thrombin cleavage siteconnected to the complete heavy chain FX (which includes FXa-PABCpeptide) showed activity after thrombin cleavage. The resulted activitydemonstrated that the FX PABC peptide was successfully conjugated to theN-terminal cysteine residue of the truncated heavy chain of FX, thecrucial N-terminal isoleucine residue was generated upon cleavage bythrombin, and the formed protein had the essential structure foractivity.

Example 6 Thrombin Activatable FX-012 with SUMO Cleavage Site

For cloning of FX-012, synthesis of the DNA sequence comprisingnucleotides from the HindIII site to the NotI site of FX-012 (Table 6)was outsourced. The DNA was subcloned into the HindIII/NotI sites ofpcDNA.

Transient expression and protein purification of FX-012 was essentiallyas described for FVII-186. Under non-reducing conditions, the major bandwith the expected molecular weight for the FX-012 was observed (FIG. 20,lane 2). Under reducing conditions, 2 major bands were observedrepresenting the desired SUMO-heavy chain FX-Fc subunit and the Fcsubunit (FIG. 20, lane 3). The LC band was not visible.

FX-012 was cleaved by a SUMO protease as follows. FX-012 (0.35 mg/mL)was incubated with 0.1 U/μL SUMO protease (Invitrogen Cat. No.12588-018), 20 mM GSH in 50 mM HEPES, 10 mM CaCl₂, pH 7.4 buffer for 24hours at room temperature. Reducing SDS-PAGE (FIG. 21, lane 2) showedalmost complete conversion of FX-012 to the desired FXHC-Fc.

For SUMO protease cleavage of FX-012 and native chemical ligation with athioester peptide, FX-012 (0.35 mg/mL) was incubated with 0.4 mM SYN470as a positive control peptide, 0.1 U/μL SUMO protease (Invitrogen Cat.No. 12588-018), 20 mM GSH in 50 mM HEPES, 10 mM CaCl₂, pH 7.4 buffer for24 hours at room temperature. Reducing SDS-PAGE (FIG. 21, lane 4) showedcomplete disappearance of the FXHC-Fc band and a single new band as theconjugate of the positive peptide control and the FXHC-Fc.

For semisynthesis of Thrombin Activatable FX-012 (TA-FX-012) by nativechemical ligation with a thioester peptide, FX-012 (0.35 mg/mL) wasincubated with 0.4 mM FX-PABC peptide (i.e.,D-Phe-Pip-Arg-PABC-IVGGQE-COSBn) (SEQ ID NO: 90), 0.1 U/μL SUMO protease(Invitrogen Cat. No. 12588-018), 20 mM GSH in 50 mM HEPES, 10 mM CaCl₂,pH 7.4 buffer for 24 hours at room temperature. Reducing SDS-PAGE (FIG.21, lane 3) showed a new band as the desired conjugate of the FX-PABCpeptide and the FXHC-Fc. Reaction mixture was placed in a 0.5 mLdialysis cassette with 10 k MWCO and dialyzed against 1 L of 10 mM Tris,250 mM NaCl, pH 8.0 containing 0.4 mM GSSG, 2 mM GSH for 24 hours at 4°C. The conjugate was further purified by rhFcRn-sepharose column asdescribed.

FXa chromogenic assay was performed after Thrombin cleavage of TA-FX-012(FIG. 22). TA-FX-012 (200 nM) was activated with Thrombin (140 nM) for20 minutes at 37° C. Hirudin was added to quench Thrombin. FXa substratewas added and reaction was monitored by measuring absorbance at 405 nm.FX-012 missing the six N-terminal amino acids was not active in thepresence of thrombin. Only TA-FX-012 with a thrombin cleavage siteconnected to the complete heavy chain FX (which includes FXa-PABCpeptide) showed activity after thrombin cleavage. The resulted activitydemonstrated that the FX PABC peptide was successfully conjugated to theN-terminal cysteine residue of the truncated heavy chain of FX, thecrucial N-terminal isoleucine residue was generated upon cleavage bythrombin, and the formed protein had the essential structure foractivity.

The present invention has been described above with the aid offunctional building blocks illustrating the implementation of specifiedfunctions and relationships thereof. The boundaries of these functionalbuilding blocks have been arbitrarily defined herein for the convenienceof the description. Alternate boundaries can be defined so long as thespecified functions and relationships thereof are appropriatelyperformed.

The foregoing description of the specific embodiments will so fullyreveal the general nature of the invention that others can, by applyingknowledge within the skill of the art, readily modify and/or adapt forvarious applications such specific embodiments, without undueexperimentation, without departing from the general concept of thepresent invention. Therefore, such adaptations and modifications areintended to be within the meaning and range of equivalents of thedisclosed embodiments, based on the teaching and guidance presentedherein. It is to be understood that the phraseology or terminologyherein is for the purpose of description and not of limitation, suchthat the terminology or phraseology of the present specification is tobe interpreted by the skilled artisan in light of the teachings andguidance.

The breadth and scope of the present invention should not be limited byany of the above-described exemplary embodiments, but should be definedonly in accordance with the following claims and their equivalents.

Tables

TABLE 2 DNA sequence of FVII-186 (SEQ ID NO: 65)    1AAGCTTGCCG CCACCATGGT CTCCCAGGCC CTCAGGCTCC TCTGCCTTCT GCTTGGGCTTTTCGAACGGC GGTGGTACCA GAGGGTCCGG GAGTCCGAGG AGACGGAAGA CGAACCCGAA   61CAGGGCTGCC TGGCTGCAGT CTTCGTAACC CAGGAGGAAG CCCACGGCGT CCTGCACCGGGTCCCGACGG ACCGACGTCA GAAGCATTGG GTCCTCCTTC GGGTGCCGCA GGACGTGGCC  121CGCCGGCGCG CCAACGCGTT CCTGGAGGAG CTGCGGCCGG GCTCCCTGGA GAGGGAGTGCGCGGCCGCGC GGTTGCGCAA GGACCTCCTC GACGCCGGCC CGAGGGACCT CTCCCTCACG  181AAGGAGGAGC AGTGCTCCTT CGAGGAGGCC CGGGAGATCT TCAAGGACGC GGAGAGGACGTTCCTCCTCG TCACGAGGAA GCTCCTCCGG GCCCTCTAGA AGTTCCTGCG CCTCTCCTGC  241AAGCTGTTCT GGATTTCTTA CAGTGATGGG GACCAGTGTG CCTCAAGTCC ATGCCAGAATTTCGACAAGA CCTAAAGAAT GTCACTACCC CTGGTCACAC GGAGTTCAGG TACGGTCTTA  301GGGGGCTCCT GCAAGGACCA GCTCCAGTCC TATATCTGCT TCTGCCTCCC TGCCTTCGAGCCCCCGAGGA CGTTCCTGGT CGAGGTCAGG ATATAGACGA AGACGGAGGG ACGGAAGCTC  361GGCCGGAACT GTGAGACGCA CAAGGATGAC CAGCTGATCT GTGTGAACGA GAACGGCGGCCCGGCCTTGA CACTCTGCGT GTTCCTACTG GTCGACTAGA CACACTTGCT CTTGCCGCCG  421TGTGAGCAGT ACTGCAGTGA CCACACGGGC ACCAAGCGCT CCTGTCGGTG CCACGAGGGGACACTCGTCA TGACGTCACT GGTGTGCCCG TGGTTCGCGA GGACAGCCAC GGTGCTCCCC  481TACTCTCTGC TGGCAGACGG GGTGTCCTGC ACACCCACAG TTGAATATCC ATGTGGAAAAATGAGAGACG ACCGTCTGCC CCACAGGACG TGTGGGTGTC AACTTATAGG TACACCTTTT  541ATACCTATTC TAGAAAAAAG AAATGCCAGC AAACCCCAAG GCCGAAAGAG GAGGAAGAGGTATGGATAAG ATCTTTTTTC TTTACGGTCG TTTGGGGTTC CGGCTTTCTC CTCCTTCTCC  601GGTGGCGGCG GATCAGGTGG GGGTGGATCA GGCGGTGGAG GTTCCCTGCA GGACTCAGAACCACCGCCGC CTAGTCCACC CCCACCTAGT CCGCCACCTC CAAGGGACGT CCTGAGTCTT  661GTCAATCAAG AAGCTAAGCC AGAGGTCAAG CCAGAAGTCA AGCCTGAGAC TCACATCAATCAGTTAGTTC TTCGATTCGG TCTCCAGTTC GGTCTTCAGT TCGGACTCTG AGTGTAGTTA  721TTAAAGGTGT CCGATGGATC TTCAGAGATC TTCTTCAAGA TCAAAAAGAC CACTCCTTTAAATTTCCACA GGCTACCTAG AAGTCTCTAG AAGAAGTTCT AGTTTTTCTG GTGAGGAAAT  781AGAAGGCTGA TGGAAGCGTT CGCTAAAAGA CAGGGTAAGG AAATGGACTC CTTAAGATTCTCTTCCGACT ACCTTCGCAA GCGATTTTCT GTCCCATTCC TTTACCTGAG GAATTCTAAG  841TTGTACGACG GTATTAGAAT TCAAGCTGAT CAGGCCCCTG AAGATTTGGA CATGGAGGATAACATGCTGC CATAATCTTA AGTTCGACTA GTCCGGGGAC TTCTAAACCT GTACCTCCTA  901AACGATATTA TTGAGGCTCA CCGCGAACAG ATTGGAGGTT GCCCCAAAGG GGAGTGTCCATTGCTATAAT AACTCCGAGT GGCGCTTGTC TAACCTCCAA CGGGGTTTCC CCTCACAGGT  961TGGCAGGTCC TGTTGTTGGT GAATGGAGCT CAGTTGTGTG GGGGGACCCT GATCAACACCACCGTCCAGG ACAACAACCA CTTACCTCGA GTCAACACAC CCCCCTGGGA CTAGTTGTGG 1021ATCTGGGTGG TCTCCGCGGC CCACTGTTTC GACAAAATCA AGAACTGGAG GAACCTGATCTAGACCCACC AGAGGCGCCG GGTGACAAAG CTGTTTTAGT TCTTGACCTC CTTGGACTAG 1081GCGGTGCTGG GCGAGCACGA CCTCAGCGAG CACGACGGGG ATGAGCAGAG CCGGCGGGTGCGCCACGACC CGCTCGTGCT GGAGTCGCTC GTGCTGCCCC TACTCGTCTC GGCCGCCCAC 1141GCGCAGGTCA TCATCCCCAG CACGTACGTC CCGGGCACCA CCAACCACGA CATCGCGCTGCGCGTCCAGT AGTAGGGGTC GTGCATGCAG GGCCCGTGGT GGTTGGTGCT GTAGCGCGAC 1201CTCCGCCTGC ACCAGCCCGT GGTCCTCACT GACCATGTGG TGCCCCTCTG CCTGCCCGAAGAGGCGGACG TGGTCGGGCA CCAGGAGTGA CTGGTACACC ACGGGGAGAC GGACGGGCTT 1261CGGACGTTCT CTGAGAGGAC GCTGGCCTTC GTGCGCTTCT CATTGGTCAG CGGCTGGGGCGCCTGCAAGA GACTCTCCTG CGACCGGAAG CACGCGAAGA GTAACCAGTC GCCGACCCCG 1321CAGCTGCTGG ACCGTGGCGC CACGGCCCTG GAGCTCATGG TCCTCAACGT GCCCCGGCTGGTCGACGACC TGGCACCGCG GTGCCGGGAC CTCGAGTACC AGGAGTTGCA CGGGGCCGAC 1381ATGACCCAGG ACTGCCTGCA GCAGTCACGG AAGGTGGGAG ACTCCCCAAA TATCACGGAGTACTGGGTCC TGACGGACGT CGTCAGTGCC TTCCACCCTC TGAGGGGTTT ATAGTGCCTC 1441TACATGTTCT GTGCCGGCTA CTCGGATGGC AGCAAGGACT CCTGCAAGGG GGACAGTGGAATGTACAAGA CACGGCCGAT GAGCCTACCG TCGTTCCTGA GGACGTTCCC CCTGTCACCT 1501GGCCCACATG CCACCCACTA CCGGGGCACG TGGTACCTGA CGGGCATCGT CAGCTGGGGCCCGGGTGTAC GGTGGGTGAT GGCCCCGTGC ACCATGGACT GCCCGTAGCA GTCGACCCCG 1561CAGGGCTGCG CAACCGTGGG CCACTTTGGG GTGTACACCA GGGTCTCCCA GTACATCGAGGTCCCGACGC GTTGGCACCC GGTGAAACCC CACATGTGGT CCCAGAGGGT CATGTAGCTC 1621TGGCTGCAAA AGCTCATGCG CTCAGAGCCA CGCCCAGGAG TCCTCCTGCG AGCCCCATTTACCGACGTTT TCGAGTACGC GAGTCTCGGT GCGGGTCCTC AGGAGGACGC TCGGGGTAAA 1681CCCGGTGGCG GTGGCTCCGG CGGAGGTGGG TCCGGTGGCG GCGGATCAGG TGGGGGTGGAGGGCCACCGC CACCGAGGCC GCCTCCACCC AGGCCACCGC CGCCTAGTCC ACCCCCACCT 1741TCAGGCGGTG GAGGTTCCGG TGGCGGGGGA TCCGACAAAA CTCACACATG CCCACCGTGCAGTCCGCCAC CTCCAAGGCC ACCGCCCCCT AGGCTGTTTT GAGTGTGTAC GGGTGGCACG 1801CCAGCTCCGG AACTCCTGGG CGGACCGTCA GTCTTCCTCT TCCCCCCAAA ACCCAAGGACGGTCGAGGCC TTGAGGACCC GCCTGGCAGT CAGAAGGAGA AGGGGGGTTT TGGGTTCCTG 1861ACCCTCATGA TCTCCCGGAC CCCTGAGGTC ACATGCGTGG TGGTGGACGT GAGCCACGAATGGGAGTACT AGAGGGCCTG GGGACTCCAG TGTACGCACC ACCACCTGCA CTCGGTGCTT 1921GACCCTGAGG TCAAGTTCAA CTGGTACGTG GACGGCGTGG AGGTGCATAA TGCCAAGACACTGGGACTCC AGTTCAAGTT GACCATGCAC CTGCCGCACC TCCACGTATT ACGGTTCTGT 1981AAGCCGCGGG AGGAGCAGTA CAACAGCACG TACCGTGTGG TCAGCGTCCT CACCGTCCTGTTCGGCGCCC TCCTCGTCAT GTTGTCGTGC ATGGCACACC AGTCGCAGGA GTGGCAGGAC 2041CACCAGGACT GGCTGAATGG CAAGGAGTAC AAGTGCAAGG TCTCCAACAA AGCCCTCCCAGTGGTCCTGA CCGACTTACC GTTCCTCATG TTCACGTTCC AGAGGTTGTT TCGGGAGGGT 2101GCCCCCATCG AGAAAACCAT CTCCAAAGCC AAAGGGCAGC CCCGAGAACC ACAGGTGTACCGGGGGTAGC TCTTTTGGTA GAGGTTTCGG TTTCCCGTCG GGGCTCTTGG TGTCCACATG 2161ACCCTGCCCC CATCCCGGGA TGAGCTGACC AAGAACCAGG TCAGCCTGAC CTGCCTGGTCTGGGACGGGG GTAGGGCCCT ACTCGACTGG TTCTTGGTCC AGTCGGACTG GACGGACCAG 2221AAAGGCTTCT ATCCCAGCGA CATCGCCGTG GAGTGGGAGA GCAATGGGCA GCCGGAGAACTTTCCGAAGA TAGGGTCGCT GTAGCGGCAC CTCACCCTCT CGTTACCCGT CGGCCTCTTG 2281AACTACAAGA CCACGCCTCC CGTGTTGGAC TCCGACGGCT CCTTCTTCCT CTACAGCAAGTTGATGTTCT GGTGCGGAGG GCACAACCTG AGGCTGCCGA GGAAGAAGGA GATGTCGTTC 2341CTCACCGTGG ACAAGAGCAG GTGGCAGCAG GGGAACGTCT TCTCATGCTC CGTGATGCATGAGTGGCACC TGTTCTCGTC CACCGTCGTC CCCTTGCAGA AGAGTACGAG GCACTACGTA 2401GAGGCTCTGC ACAACCACTA CACGCAGAAG AGCCTCTCCC TGTCTCCGGG TAAAGGTGGCCTCCGAGACG TGTTGGTGAT GTGCGTCTTC TCGGAGAGGG ACAGAGGCCC ATTTCCACCG 2461GGCGGATCAG GTGGGGGTGG ATCAGGCGGT GGAGGTTCCG GTGGCGGGGG ATCAGACAAACCGCCTAGTC CACCCCCACC TAGTCCGCCA CCTCCAAGGC CACCGCCCCC TAGTCTGTTT 2521ACTCACACAT GCCCACCGTG CCCAGCACCT GAACTCCTGG GAGGACCGTC AGTCTTCCTCTGAGTGTGTA CGGGTGGCAC GGGTCGTGGA CTTGAGGACC CTCCTGGCAG TCAGAAGGAG 2581TTCCCCCCAA AACCCAAGGA CACCCTCATG ATCTCCCGGA CCCCTGAGGT CACATGCGTGAAGGGGGGTT TTGGGTTCCT GTGGGAGTAC TAGAGGGCCT GGGGACTCCA GTGTACGCAC 2641GTGGTGGACG TGAGCCACGA AGACCCTGAG GTCAAGTTCA ACTGGTACGT GGACGGCGTGCACCACCTGC ACTCGGTGCT TCTGGGACTC CAGTTCAAGT TGACCATGCA CCTGCCGCAC 2701GAGGTGCATA ATGCCAAGAC AAAGCCGCGG GAGGAGCAGT ACAACAGCAC GTACCGTGTGCTCCACGTAT TACGGTTCTG TTTCGGCGCC CTCCTCGTCA TGTTGTCGTG CATGGCACAC 2761GTCAGCGTCC TCACCGTCCT GCACCAGGAC TGGCTGAATG GCAAGGAGTA CAAGTGCAAGCAGTCGCAGG AGTGGCAGGA CGTGGTCCTG ACCGACTTAC CGTTCCTCAT GTTCACGTTC 2821GTCTCCAACA AAGCCCTCCC AGCCCCCATC GAGAAAACCA TCTCCAAAGC CAAAGGGCAGCAGAGGTTGT TTCGGGAGGG TCGGGGGTAG CTCTTTTGGT AGAGGTTTCG GTTTCCCGTC 2881CCCCGAGAAC CACAGGTGTA CACCCTGCCC CCATCCCGCG ATGAGCTGAC CAAGAACCAGGGGGCTCTTG GTGTCCACAT GTGGGACGGG GGTAGGGCGC TACTCGACTG GTTCTTGGTC 2941GTCAGCCTGA CCTGCCTGGT CAAAGGCTTC TATCCCAGCG ACATCGCCGT GGAGTGGGAGCAGTCGGACT GGACGGACCA GTTTCCGAAG ATAGGGTCGC TGTAGCGGCA CCTCACCCTC 3001AGCAATGGGC AGCCGGAGAA CAACTACAAG ACCACGCCTC CCGTGTTGGA CTCCGACGGCTCGTTACCCG TCGGCCTCTT GTTGATGTTC TGGTGCGGAG GGCACAACCT GAGGCTGCCG 3061TCCTTCTTCC TCTACAGCAA GCTCACCGTG GACAAGAGCA GGTGGCAGCA GGGGAACGTCAGGAAGAAGG AGATGTCGTT CGAGTGGCAC CTGTTCTCGT CCACCGTCGT CCCCTTGCAG 3121TTCTCATGCT CCGTGATGCA TGAGGCTCTG CACAACCACT ACACGCAGAA GAGCCTCTCCAAGAGTACGA GGCACTACGT ACTCCGAGAC GTGTTGGTGA TGTGCGTCTT CTCGGAGAGG 3181CTGTCTCCGG GTAAATGAGA ATTC GACAGAGGCC CATTTACTCT TAAG

TABLE 3FVII-186 amino acid sequence. Signal sequence is shown in dotted underline,propeptide is double underlined, linker with proprotein convertaseprocessing sites connecting the FVII light chain to SUMO is underlined,SUMO sequence is in dashed underline, linker region connectingFVII to Fc region is in italic, and linker connecting the twoFc fragments is shown in bold (SEQ ID NO: 66)    1

  61 SFEEAREIFK DAERTKLFWI SYSDGDQCAS SPCQNGGSCK DQLQSYICFC LPAFEGRNCE 121 THKDDQLICV NENGGCEQYC SDHTGTKRSC RCHEGYSLLA DGVSCTPTVE YPCGKIPILE 181

 241

 301

 361 HDLSEHDGDE QSRRVAQVII PSTYVPGTTN HDIALLRLHQ PVVLTDHVVP LCLPERTFSE 421 RTLAFVRFSL VSGWGQLLDR GATALELMVL NVPRLMTQDC LQQSRKVGDS PNITEYMFCA 481 GYSDGSKDSC KGDSGGPHAT HYRGTWYLTG IVSWGQGCAT VGHFGVYTRV SQYIEWLQKL 541 MRSEPRPGVL LRAPFPGGGG SGGGGSGGGG SGGGGSGGGG SGGGGSDKTH TCPPCPAPEL 601 LGGPSVFLFP PKPKDTLMIS RTPEVTCVVV DVSHEDPEVK FNWYVDGVEV HNAKTKPREE 661 QYNSTYRVVS VLTVLHQDWL NGKEYKCKVS NKALPAPIEK TISKAKGQPR EPQVYTLPPS 721 RDELTKNQVS LTCLVKGFYP SDIAVEWESN GQPENNYKTT PPVLDSDGSF FLYSKLTVDK 781 SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PGKGGGGSGG GGSGGGGSGG GGSDKTHTCP 841 PCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA 901 KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ 961 VYTLPPSRDE LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY1021 SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK*

TABLE 4 DNA sequence of FX-011 (SEQ ID NO: 67)    1AAGCTTATGG GTCGTCCACT GCACCTCGTC CTGCTCAGTG CCTCCCTGGC TGGCCTCCTGTTCGAATACC CAGCAGGTGA CGTGGAGCAG GACGAGTCAC GGAGGGACCG ACCGGAGGAC   61CTGCTCGGGG AAAGTCTGTT CATCCGCAGG GAGCAGGCCA ACAACATCCT GGCGAGGGTCGACGAGCCCC TTTCAGACAA GTAGGCGTCC CTCGTCCGGT TGTTGTAGGA CCGCTCCCAG  121AGGAGGGCCA ATTCCTTTCT TGAAGAGATG AAGAAAGGAC ACCTCGAAAG AGAGTGCATGTCCTCCCGGT TAAGGAAAGA ACTTCTCTAC TTCTTTCCTG TGGAGCTTTC TCTCACGTAC  181GAAGAGACCT GCTCATACGA AGAGGCCCGC GAGGTCTTTG AGGACAGCGA CAAGACGAATCTTCTCTGGA CGAGTATGCT TCTCCGGGCG CTCCAGAAAC TCCTGTCGCT GTTCTGCTTA  241GAATTCTGGA ATAAATACAA AGATGGCGAC CAGTGTGAGA CCAGTCCTTG CCAGAACCAGCTTAAGACCT TATTTATGTT TCTACCGCTG GTCACACTCT GGTCAGGAAC GGTCTTGGTC  301GGCAAATGTA AAGACGGCCT CGGGGAATAC ACCTGCACCT GTTTAGAAGG ATTCGAAGGCCCGTTTACAT TTCTGCCGGA GCCCCTTATG TGGACGTGGA CAAATCTTCC TAAGCTTCCG  361AAAAACTGTG AATTATTCAC ACGGAAGCTC TGCAGCCTGG ACAACGGGGA CTGTGACCAGTTTTTGACAC TTAATAAGTG TGCCTTCGAG ACGTCGGACC TGTTGCCCCT GACACTGGTC  421TTCTGCCACG AGGAACAGAA CTCTGTGGTG TGCTCCTGCG CCCGCGGGTA CACCCTGGCTAAGACGGTGC TCCTTGTCTT GAGACACCAC ACGAGGACGC GGGCGCCCAT GTGGGACCGA  481GACAACGGCA AGGCCTGCAT TCCCACAGGG CCCTACCCCT GTGGGAAACA GACCCTGGAACTGTTGCCGT TCCGGACGTA AGGGTGTCCC GGGATGGGGA CACCCTTTGT CTGGGACCTT  541CGCAGGAAGA GGTCAGTGGC CCAGGCCACC AGCAGCAGCG GGGAGGCCCC TGACAGCATCGCGTCCTTCT CCAGTCACCG GGTCCGGTGG TCGTCGTCGC CCCTCCGGGG ACTGTCGTAG  601ACATGGAAGC CATATGATGC AGCCGACCTG GACCCCACCG AGAACCCCTT CGACCTGCTTTGTACCTTCG GTATACTACG TCGGCTGGAC CTGGGGTGGC TCTTGGGGAA GCTGGACGAA  661GACTTCAACC AGACGCAGCC TGAGAGGGGC GACAACAACG GTGGCGGCGG ATCAGGTGGGCTGAAGTTGG TCTGCGTCGG ACTCTCCCCG CTGTTGTTGC CACCGCCGCC TAGTCCACCC  721GGTGGATCAG GCGGTGGAGG TTCCGGTGGC GGGGGATCCA GGAAGAGGAG GAAGAGGTGCCCACCTAGTC CGCCACCTCC AAGGCCACCG CCCCCTAGGT CCTTCTCCTC CTTCTCCACG  781AAGGACGGGG AGTGTCCCTG GCAGGCCCTG CTCATCAATG AGGAAAACGA GGGTTTTTGTTTCCTGCCCC TCACAGGGAC CGTCCGGGAC GAGTAGTTAC TCCTTTTGCT CCCAAAAACA  841GGAGGTACCA TTCTGAGCGA GTTCTACATC CTAACGGCAG CCCACTGTCT CTACCAAGCCCCTCCATGGT AAGACTCGCT CAAGATGTAG GATTGCCGTC GGGTGACAGA GATGGTTCGG  901AAGAGATTCA AGGTGAGGGT AGGGGACCGG AACACGGAGC AGGAGGAGGG CGGTGAGGCGTTCTCTAAGT TCCACTCCCA TCCCCTGGCC TTGTGCCTCG TCCTCCTCCC GCCACTCCGC  961GTGCACGAGG TGGAGGTGGT CATCAAGCAC AACCGGTTCA CAAAGGAGAC CTATGACTTCCACGTGCTCC ACCTCCACCA GTAGTTCGTG TTGGCCAAGT GTTTCCTCTG GATACTGAAG 1021GACATCGCCG TGCTCCGGCT CAAGACCCCC ATCACCTTCC GCATGAACGT GGCGCCTGCCCTGTAGCGGC ACGAGGCCGA GTTCTGGGGG TAGTGGAAGG CGTACTTGCA CCGCGGACGG 1081TGCCTCCCCG AGCGTGACTG GGCCGAGTCC ACGCTGATGA CGCAGAAGAC GGGGATTGTGACGGAGGGGC TCGCACTGAC CCGGCTCAGG TGCGACTACT GCGTCTTCTG CCCCTAACAC 1141AGCGGCTTCG GGCGCACCCA CGAGAAGGGC CGGCAGTCCA CCAGGCTCAA GATGCTGGAGTCGCCGAAGC CCGCGTGGGT GCTCTTCCCG GCCGTCAGGT GGTCCGAGTT CTACGACCTC 1201GTGCCCTACG TGGACCGCAA CAGCTGCAAG CTGTCCAGCA GCTTCATCAT CACCCAGAACCACGGGATGC ACCTGGCGTT GTCGACGTTC GACAGGTCGT CGAAGTAGTA GTGGGTCTTG 1261ATGTTCTGTG CCGGCTACGA CACCAAGCAG GAGGATGCCT GCCAGGGGGA CAGCGGGGGCTACAAGACAC GGCCGATGCT GTGGTTCGTC CTCCTACGGA CGGTCCCCCT GTCGCCCCCG 1321CCGCACGTCA CCCGCTTCAA GGACACCTAC TTCGTGACAG GCATCGTCAG CTGGGGAGAGGGCGTGCAGT GGGCGAAGTT CCTGTGGATG AAGCACTGTC CGTAGCAGTC GACCCCTCTC 1381GGCTGTGCCC GTAAGGGGAA GTACGGGATC TACACCAAGG TCACCGCCTT CCTCAAGTGGCCGACACGGG CATTCCCCTT CATGCCCTAG ATGTGGTTCC AGTGGCGGAA GGAGTTCACC 1441ATCGACAGGT CCATGAAAAC CAGGGGCTTG CCCAAGGCCA AGAGCCATGC CCCGGAGGTCTAGCTGTCCA GGTACTTTTG GTCCCCGAAC GGGTTCCGGT TCTCGGTACG GGGCCTCCAG 1501ATAACGTCCT CTCCATTAAA AGAAGACCAA GTAGATCCGC GGCTCATTGA TGGTAAGGACTATTGCAGGA GAGGTAATTT TCTTCTGGTT CATCTAGGCG CCGAGTAACT ACCATTCCTG 1561AAAACTCACA CATGCCCACC GTGCCCAGCT CCGGAACTCC TGGGAGGACC GTCAGTCTTCTTTTGAGTGT GTACGGGTGG CACGGGTCGA GGCCTTGAGG ACCCTCCTGG CAGTCAGAAG 1621CTCTTCCCCC CAAAACCCAA GGACACCCTC ATGATCTCCC GGACCCCTGA GGTCACATGCGAGAAGGGGG GTTTTGGGTT CCTGTGGGAG TACTAGAGGG CCTGGGGACT CCAGTGTACG 1681GTGGTGGTGG ACGTGAGCCA CGAAGACCCT GAGGTCAAGT TCAACTGGTA CGTGGACGGCCACCACCACC TGCACTCGGT GCTTCTGGGA CTCCAGTTCA AGTTGACCAT GCACCTGCCG 1741GTGGAGGTGC ATAATGCCAA GACAAAGCCG CGGGAGGAGC AGTACAACAG CACGTACCGTCACCTCCACG TATTACGGTT CTGTTTCGGC GCCCTCCTCG TCATGTTGTC GTGCATGGCA 1801GTGGTCAGCG TCCTCACCGT CCTGCACCAG GACTGGCTGA ATGGCAAGGA GTACAAGTGCCACCAGTCGC AGGAGTGGCA GGACGTGGTC CTGACCGACT TACCGTTCCT CATGTTCACG 1861AAGGTCTCCA ACAAAGCCCT CCCAGCCCCC ATCGAGAAAA CCATCTCCAA AGCCAAAGGGTTCCAGAGGT TGTTTCGGGA GGGTCGGGGG TAGCTCTTTT GGTAGAGGTT TCGGTTTCCC 1921CAGCCCCGAG AACCACAGGT GTACACCCTG CCCCCATCCC GGGATGAGCT GACCAAGAACGTCGGGGCTC TTGGTGTCCA CATGTGGGAC GGGGGTAGGG CCCTACTCGA CTGGTTCTTG 1981CAGGTCAGCC TGACCTGCCT GGTCAAAGGC TTCTATCCCA GCGACATCGC CGTGGAGTGGGTCCAGTCGG ACTGGACGGA CCAGTTTCCG AAGATAGGGT CGCTGTAGCG GCACCTCACC 2041GAGAGCAATG GGCAGCCGGA GAACAACTAC AAGACCACGC CTCCCGTGTT GGACTCCGACCTCTCGTTAC CCGTCGGCCT CTTGTTGATG TTCTGGTGCG GAGGGCACAA CCTGAGGCTG 2101GGCTCCTTCT TCCTCTACAG CAAGCTCACC GTCGACAAGA GCAGGTGGCA GCAGGGGAACCCGAGGAAGA AGGAGATGTC GTTCGAGTGG CAGCTGTTCT CGTCCACCGT CGTCCCCTTG 2161GTCTTCTCAT GCTCCGTGAT GCATGAGGCT CTGCACAACC ACTACACGCA GAAGAGCCTCCAGAAGAGTA CGAGGCACTA CGTACTCCGA GACGTGTTGG TGATGTGCGT CTTCTCGGAG 2221TCCCTGTCTC CGGGTAAACG GCGCCGCCGG AGCGGTGGCG GCGGATCAGG TGGGGGTGGAAGGGACAGAG GCCCATTTGC CGCGGCGGCC TCGCCACCGC CGCCTAGTCC ACCCCCACCT 2281TCAGGCGGTG GAGGTTCCGG TGGCGGGGGA TCCAGGAAGA GGAGGAAGAG GGACAAAACTAGTCCGCCAC CTCCAAGGCC ACCGCCCCCT AGGTCCTTCT CCTCCTTCTC CCTGTTTTGA 2341CACACATGCC CACCGTGCCC AGCACCGGAA CTCCTGGGCG GACCGTCAGT CTTCCTCTTCGTGTGTACGG GTGGCACGGG TCGTGGCCTT GAGGACCCGC CTGGCAGTCA GAAGGAGAAG 2401CCCCCAAAAC CCAAGGACAC CCTCATGATC TCCCGGACCC CTGAGGTCAC ATGCGTGGTGGGGGGTTTTG GGTTCCTGTG GGAGTACTAG AGGGCCTGGG GACTCCAGTG TACGCACCAC 2461GTGGACGTGA GCCACGAAGA CCCTGAGGTC AAGTTCAACT GGTACGTGGA CGGCGTGGAGCACCTGCACT CGGTGCTTCT GGGACTCCAG TTCAAGTTGA CCATGCACCT GCCGCACCTC 2521GTGCATAATG CCAAGACAAA GCCGCGGGAG GAGCAGTACA ACAGCACGTA CCGTGTGGTCCACGTATTAC GGTTCTGTTT CGGCGCCCTC CTCGTCATGT TGTCGTGCAT GGCACACCAG 2581AGCGTCCTCA CCGTCCTGCA CCAGGACTGG CTGAATGGCA AGGAGTACAA GTGCAAGGTCTCGCAGGAGT GGCAGGACGT GGTCCTGACC GACTTACCGT TCCTCATGTT CACGTTCCAG 2641TCCAACAAAG CCCTCCCAGC CCCCATCGAG AAAACCATCT CCAAAGCCAA AGGGCAGCCCAGGTTGTTTC GGGAGGGTCG GGGGTAGCTC TTTTGGTAGA GGTTTCGGTT TCCCGTCGGG 2701CGAGAACCAC AGGTGTACAC CCTGCCCCCA TCCCGGGATG AGCTGACCAA GAACCAGGTCGCTCTTGGTG TCCACATGTG GGACGGGGGT AGGGCCCTAC TCGACTGGTT CTTGGTCCAG 2761AGCCTGACCT GCCTGGTCAA AGGCTTCTAT CCCAGCGACA TCGCCGTGGA GTGGGAGAGCTCGGACTGGA CGGACCAGTT TCCGAAGATA GGGTCGCTGT AGCGGCACCT CACCCTCTCG 2821AATGGGCAGC CGGAGAACAA CTACAAGACC ACGCCTCCCG TGTTGGACTC CGACGGCTCCTTACCCGTCG GCCTCTTGTT GATGTTCTGG TGCGGAGGGC ACAACCTGAG GCTGCCGAGG 2881TTCTTCCTCT ACAGCAAGCT CACCGTGGAC AAGAGCAGGT GGCAGCAGGG GAACGTCTTCAAGAAGGAGA TGTCGTTCGA GTGGCACCTG TTCTCGTCCA CCGTCGTCCC CTTGCAGAAG 2941TCATGCTCCG TGATGCATGA GGCTCTGCAC AACCACTACA CGCAGAAGAG CCTCTCCCTGAGTACGAGGC ACTACGTACT CCGAGACGTG TTGGTGATGT GCGTCTTCTC GGAGAGGGAC 3001TCTCCGGGTA AATGAGCGGC CGC AGAGGCCCAT TTACTCGCCG GCG

TABLE 5 FX-011 amino acid sequence. Signal sequence is shown in dottedunderline, propeptide is double underlined, linker region connecting FXactivation peptide to FX heavy chain is underlined, and linker withproprotein convertase processing sites connecting the two Fcfragments is shown in bold (SEQ ID NO: 68)   1

 61 TCSYEEAREV FEDSDKTNEF WNKYKDGDQC ETSPCQNQGK CKDGLGEYTC TCLEGFEGKN121 CELFTRKLCS LDNGDCDQFC HEEQNSVVCS CARGYTLADN GKACIPTGPY PCGKQTLERR181 KRSVAQATSS SGEAPDSITW KPYDAADLDP TENPFDLLDF NQTQPERGDN NGGGGSGGGG241 SGGGGSGGGG SRKRRKRCKD GECPWQALLI NEENEGFCGG TILSEFYILT AAHCLYQAKR301 FKVRVGDRNT EQEEGGEAVH EVEVVIKHNR FTKETYDFDI AVLRLKTPIT FRMNVAPACL361 PERDWAESTL MTQKTGIVSG FGRTHEKGRQ STRLKMLEVP YVDRNSCKLS SSFIITQNMF421 CAGYDTKQED ACQGDSGGPH VTRFKDTYFV TGIVSWGEGC ARKGKYGIYT KVTAFLKWID481 RSMKTRGLPK AKSHAPEVIT SSPLKEDQVD PRLIDGKDKT HTCPPCPAPE LLGGPSVFLF541 PPKPKDTLMI SRTPEVTCVV VDVSHEDPEV KFNWYVDGVE VHNAKTKPRE EQYNSTYRVV601 SVLTVLHQDW LNGKEYKCKV SNKALPAPIE KTISKAKGQP REPQVYTLPP SRDELTKNQV661 SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSKLTVD KSRWQQGNVF721 SCSVMHEALH NHYTQKSLSL SPGKRRRRSG GGGSGGGGSG GGGSGGGGSR KRRKRDKTHT781 CPPCPAPELL GGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSHEDPEVKF NWYVDGVEVH841 NAKTKPREEQ YNSTYRVVSV LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE901 PQVYTLPPSR DELTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF961 LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GK*

TABLE 6 DNA sequence of FX-012 (SEQ ID NO: 69)    1AAGCTTATGG GTCGTCCACT GCACCTCGTC CTGCTCAGTG CCTCCCTGGC TGGCCTCCTGTTCGAATACC CAGCAGGTGA CGTGGAGCAG GACGAGTCAC GGAGGGACCG ACCGGAGGAC   61CTGCTCGGGG AAAGTCTGTT CATCCGCAGG GAGCAGGCCA ACAACATCCT GGCGAGGGTCGACGAGCCCC TTTCAGACAA GTAGGCGTCC CTCGTCCGGT TGTTGTAGGA CCGCTCCCAG  121AGGAGGGCCA ATTCCTTTCT TGAAGAGATG AAGAAAGGAC ACCTCGAAAG AGAGTGCATGTCCTCCCGGT TAAGGAAAGA ACTTCTCTAC TTCTTTCCTG TGGAGCTTTC TCTCACGTAC  181GAAGAGACCT GCTCATACGA AGAGGCCCGC GAGGTCTTTG AGGACAGCGA CAAGACGAATCTTCTCTGGA CGAGTATGCT TCTCCGGGCG CTCCAGAAAC TCCTGTCGCT GTTCTGCTTA  241GAATTCTGGA ATAAATACAA AGATGGCGAC CAGTGTGAGA CCAGTCCTTG CCAGAACCAGCTTAAGACCT TATTTATGTT TCTACCGCTG GTCACACTCT GGTCAGGAAC GGTCTTGGTC  301GGCAAATGTA AAGACGGCCT CGGGGAATAC ACCTGCACCT GTTTAGAAGG ATTCGAAGGCCCGTTTACAT TTCTGCCGGA GCCCCTTATG TGGACGTGGA CAAATCTTCC TAAGCTTCCG  361AAAAACTGTG AATTATTCAC ACGGAAGCTC TGCAGCCTGG ACAACGGGGA CTGTGACCAGTTTTTGACAC TTAATAAGTG TGCCTTCGAG ACGTCGGACC TGTTGCCCCT GACACTGGTC  421TTCTGCCACG AGGAACAGAA CTCTGTGGTG TGCTCCTGCG CCCGCGGGTA CACCCTGGCTAAGACGGTGC TCCTTGTCTT GAGACACCAC ACGAGGACGC GGGCGCCCAT GTGGGACCGA  481GACAACGGCA AGGCCTGCAT TCCCACAGGG CCCTACCCCT GTGGGAAACA GACCCTGGAACTGTTGCCGT TCCGGACGTA AGGGTGTCCC GGGATGGGGA CACCCTTTGT CTGGGACCTT  541CGCAGGAAGA GGGGTGGGGG TGGATCAGGC GGTGGAGGTT CCGGTGGCGG GGGATCCTCCGCGTCCTTCT CCCCACCCCC ACCTAGTCCG CCACCTCCAA GGCCACCGCC CCCTAGGAGG  601CTGCAGGACT CAGAAGTCAA TCAAGAAGCT AAGCCAGAGG TCAAGCCAGA AGTCAAGCCTGACGTCCTGA GTCTTCAGTT AGTTCTTCGA TTCGGTCTCC AGTTCGGTCT TCAGTTCGGA  661GAGACTCACA TCAATTTAAA GGTGTCCGAT GGATCTTCAG AGATCTTCTT CAAGATCAAACTCTGAGTGT AGTTAAATTT CCACAGGCTA CCTAGAAGTC TCTAGAAGAA GTTCTAGTTT  721AAGACCACTC CTTTAAGAAG GCTGATGGAA GCGTTCGCTA AAAGACAGGG TAAGGAAATGTTCTGGTGAG GAAATTCTTC CGACTACCTT CGCAAGCGAT TTTCTGTCCC ATTCCTTTAC  781GACTCCTTAA GATTCTTGTA CGACGGTATT AGAATTCAAG CTGATCAGGC CCCTGAAGATCTGAGGAATT CTAAGAACAT GCTGCCATAA TCTTAAGTTC GACTAGTCCG GGGACTTCTA  841TTGGACATGG AGGATAACGA TATTATTGAG GCTCACCGCG AACAGATTGG AGGTTGCAAGAACCTGTACC TCCTATTGCT ATAATAACTC CGAGTGGCGC TTGTCTAACC TCCAACGTTC  901GACGGGGAGT GTCCCTGGCA GGCCCTGCTC ATCAATGAGG AAAACGAGGG TTTTTGTGGACTGCCCCTCA CAGGGACCGT CCGGGACGAG TAGTTACTCC TTTTGCTCCC AAAAACACCT  961GGTACCATTC TGAGCGAGTT CTACATCCTA ACGGCAGCCC ACTGTCTCTA CCAAGCCAAGCCATGGTAAG ACTCGCTCAA GATGTAGGAT TGCCGTCGGG TGACAGAGAT GGTTCGGTTC 1021AGATTCAAGG TGAGGGTAGG GGACCGGAAC ACGGAGCAGG AGGAGGGCGG TGAGGCGGTGTCTAAGTTCC ACTCCCATCC CCTGGCCTTG TGCCTCGTCC TCCTCCCGCC ACTCCGCCAC 1081CACGAGGTGG AGGTGGTCAT CAAGCACAAC CGGTTCACAA AGGAGACCTA TGACTTCGACGTGCTCCACC TCCACCAGTA GTTCGTGTTG GCCAAGTGTT TCCTCTGGAT ACTGAAGCTG 1141ATCGCCGTGC TCCGGCTCAA GACCCCCATC ACCTTCCGCA TGAACGTGGC GCCTGCCTGCTAGCGGCACG AGGCCGAGTT CTGGGGGTAG TGGAAGGCGT ACTTGCACCG CGGACGGACG 1201CTCCCCGAGC GTGACTGGGC CGAGTCCACG CTGATGACGC AGAAGACGGG GATTGTGAGCGAGGGGCTCG CACTGACCCG GCTCAGGTGC GACTACTGCG TCTTCTGCCC CTAACACTCG 1261GGCTTCGGGC GCACCCACGA GAAGGGCCGG CAGTCCACCA GGCTCAAGAT GCTGGAGGTGCCGAAGCCCG CGTGGGTGCT CTTCCCGGCC GTCAGGTGGT CCGAGTTCTA CGACCTCCAC 1321CCCTACGTGG ACCGCAACAG CTGCAAGCTG TCCAGCAGCT TCATCATCAC CCAGAACATGGGGATGCACC TGGCGTTGTC GACGTTCGAC AGGTCGTCGA AGTAGTAGTG GGTCTTGTAC 1381TTCTGTGCCG GCTACGACAC CAAGCAGGAG GATGCCTGCC AGGGGGACAG CGGGGGCCCGAAGACACGGC CGATGCTGTG GTTCGTCCTC CTACGGACGG TCCCCCTGTC GCCCCCGGGC 1441CACGTCACCC GCTTCAAGGA CACCTACTTC GTGACAGGCA TCGTCAGCTG GGGAGAGGGCGTGCAGTGGG CGAAGTTCCT GTGGATGAAG CACTGTCCGT AGCAGTCGAC CCCTCTCCCG 1501TGTGCCCGTA AGGGGAAGTA CGGGATCTAC ACCAAGGTCA CCGCCTTCCT CAAGTGGATCACACGGGCAT TCCCCTTCAT GCCCTAGATG TGGTTCCAGT GGCGGAAGGA GTTCACCTAG 1561GACAGGTCCA TGAAAACCAG GGGCTTGCCC AAGGCCAAGA GCCATGCCCC GGAGGTCATACTGTCCAGGT ACTTTTGGTC CCCGAACGGG TTCCGGTTCT CGGTACGGGG CCTCCAGTAT 1621ACGTCCTCTC CATTAAAAGA AGACCAAGTA GATCCGCGGC TCATTGATGG TAAGGACAAATGCAGGAGAG GTAATTTTCT TCTGGTTCAT CTAGGCGCCG AGTAACTACC ATTCCTGTTT 1681ACTCACACAT GCCCACCGTG CCCAGCTCCG GAACTCCTGG GAGGACCGTC AGTCTTCCTCTGAGTGTGTA CGGGTGGCAC GGGTCGAGGC CTTGAGGACC CTCCTGGCAG TCAGAAGGAG 1741TTCCCCCCAA AACCCAAGGA CACCCTCATG ATCTCCCGGA CCCCTGAGGT CACATGCGTGAAGGGGGGTT TTGGGTTCCT GTGGGAGTAC TAGAGGGCCT GGGGACTCCA GTGTACGCAC 1801GTGGTGGACG TGAGCCACGA AGACCCTGAG GTCAAGTTCA ACTGGTACGT GGACGGCGTGCACCACCTGC ACTCGGTGCT TCTGGGACTC CAGTTCAAGT TGACCATGCA CCTGCCGCAC 1861GAGGTGCATA ATGCCAAGAC AAAGCCGCGG GAGGAGCAGT ACAACAGCAC GTACCGTGTGCTCCACGTAT TACGGTTCTG TTTCGGCGCC CTCCTCGTCA TGTTGTCGTG CATGGCACAC 1921GTCAGCGTCC TCACCGTCCT GCACCAGGAC TGGCTGAATG GCAAGGAGTA CAAGTGCAAGCAGTCGCAGG AGTGGCAGGA CGTGGTCCTG ACCGACTTAC CGTTCCTCAT GTTCACGTTC 1981GTCTCCAACA AAGCCCTCCC AGCCCCCATC GAGAAAACCA TCTCCAAAGC CAAAGGGCAGCAGAGGTTGT TTCGGGAGGG TCGGGGGTAG CTCTTTTGGT AGAGGTTTCG GTTTCCCGTC 2041CCCCGAGAAC CACAGGTGTA CACCCTGCCC CCATCCCGGG ATGAGCTGAC CAAGAACCAGGGGGCTCTTG GTGTCCACAT GTGGGACGGG GGTAGGGCCC TACTCGACTG GTTCTTGGTC 2101GTCAGCCTGA CCTGCCTGGT CAAAGGCTTC TATCCCAGCG ACATCGCCGT GGAGTGGGAGCAGTCGGACT GGACGGACCA GTTTCCGAAG ATAGGGTCGC TGTAGCGGCA CCTCACCCTC 2161AGCAATGGGC AGCCGGAGAA CAACTACAAG ACCACGCCTC CCGTGTTGGA CTCCGACGGCTCGTTACCCG TCGGCCTCTT GTTGATGTTC TGGTGCGGAG GGCACAACCT GAGGCTGCCG 2221TCCTTCTTCC TCTACAGCAA GCTCACCGTC GACAAGAGCA GGTGGCAGCA GGGGAACGTCAGGAAGAAGG AGATGTCGTT CGAGTGGCAG CTGTTCTCGT CCACCGTCGT CCCCTTGCAG 2281TTCTCATGCT CCGTGATGCA TGAGGCTCTG CACAACCACT ACACGCAGAA GAGCCTCTCCAAGAGTACGA GGCACTACGT ACTCCGAGAC GTGTTGGTGA TGTGCGTCTT CTCGGAGAGG 2341CTGTCTCCGG GTAAACGGCG CCGCCGGAGC GGTGGCGGCG GATCAGGTGG GGGTGGATCAGACAGAGGCC CATTTGCCGC GGCGGCCTCG CCACCGCCGC CTAGTCCACC CCCACCTAGT 2401GGCGGTGGAG GTTCCGGTGG CGGGGGATCC AGGAAGAGGA GGAAGAGGGA CAAAACTCACCCGCCACCTC CAAGGCCACC GCCCCCTAGG TCCTTCTCCT CCTTCTCCCT GTTTTGAGTG 2461ACATGCCCAC CGTGCCCAGC ACCGGAACTC CTGGGCGGAC CGTCAGTCTT CCTCTTCCCCTGTACGGGTG GCACGGGTCG TGGCCTTGAG GACCCGCCTG GCAGTCAGAA GGAGAAGGGG 2521CCAAAACCCA AGGACACCCT CATGATCTCC CGGACCCCTG AGGTCACATG CGTGGTGGTGGGTTTTGGGT TCCTGTGGGA GTACTAGAGG GCCTGGGGAC TCCAGTGTAC GCACCACCAC 2581GACGTGAGCC ACGAAGACCC TGAGGTCAAG TTCAACTGGT ACGTGGACGG CGTGGAGGTGCTGCACTCGG TGCTTCTGGG ACTCCAGTTC AAGTTGACCA TGCACCTGCC GCACCTCCAC 2641CATAATGCCA AGACAAAGCC GCGGGAGGAG CAGTACAACA GCACGTACCG TGTGGTCAGCGTATTACGGT TCTGTTTCGG CGCCCTCCTC GTCATGTTGT CGTGCATGGC ACACCAGTCG 2701GTCCTCACCG TCCTGCACCA GGACTGGCTG AATGGCAAGG AGTACAAGTG CAAGGTCTCCCAGGAGTGGC AGGACGTGGT CCTGACCGAC TTACCGTTCC TCATGTTCAC GTTCCAGAGG 2761AACAAAGCCC TCCCAGCCCC CATCGAGAAA ACCATCTCCA AAGCCAAAGG GCAGCCCCGATTGTTTCGGG AGGGTCGGGG GTAGCTCTTT TGGTAGAGGT TTCGGTTTCC CGTCGGGGCT 2821GAACCACAGG TGTACACCCT GCCCCCATCC CGGGATGAGC TGACCAAGAA CCAGGTCAGCCTTGGTGTCC ACATGTGGGA CGGGGGTAGG GCCCTACTCG ACTGGTTCTT GGTCCAGTCG 2881CTGACCTGCC TGGTCAAAGG CTTCTATCCC AGCGACATCG CCGTGGAGTG GGAGAGCAATGACTGGACGG ACCAGTTTCC GAAGATAGGG TCGCTGTAGC GGCACCTCAC CCTCTCGTTA 2941GGGCAGCCGG AGAACAACTA CAAGACCACG CCTCCCGTGT TGGACTCCGA CGGCTCCTTCCCCGTCGGCC TCTTGTTGAT GTTCTGGTGC GGAGGGCACA ACCTGAGGCT GCCGAGGAAG 3001TTCCTCTACA GCAAGCTCAC CGTGGACAAG AGCAGGTGGC AGCAGGGGAA CGTCTTCTCAAAGGAGATGT CGTTCGAGTG GCACCTGTTC TCGTCCACCG TCGTCCCCTT GCAGAAGAGT 3061TGCTCCGTGA TGCATGAGGC TCTGCACAAC CACTACACGC AGAAGAGCCT CTCCCTGTCTACGAGGCACT ACGTACTCCG AGACGTGTTG GTGATGTGCG TCTTCTCGGA GAGGGACAGA 3121CCGGGTAAAT GAGCGGCCGC GGCCCATTTA CTCGCCGGCG

TABLE 7 FX-012 amino acid sequence. Signal sequence is shown in dottedunderline, propeptide is double underlined, linker region connectingFX light chain to SUMO is underlined, SUMO sequence is in dashedunderline, and linker with proprotein convertase processing sitesconnecting the two Fc fragments is shown in bold (SEQ ID NO: 70)    1

  61 TCSYEEAREV FEDSDKTNEF WNKYKDGDQC ETSPCQNQGK CKDGLGEYTC TCLEGFEGKN 121 CELFTRKLCS LDNGDCDQFC HEEQNSVVCS CARGYTLADN GKACIPTGPY PCGKQTLERR 181

 241

 301 ECPWQALLIN EENEGFCGGT ILSEFYILTA AHCLYQAKRF KVRVGDRNTE QEEGGEAVHE 361 VEVVIKHNRF TKETYDFDIA VLRLKTPITF RMNVAPACLP ERDWAESTLM TQKTGIVSGF 421 GRTHEKGRQS TRLKMLEVPY VDRNSCKLSS SFIITQNMFC AGYDTKQEDA CQGDSGGPHV 481 TRFKDTYFVT GIVSWGEGCA RKGKYGIYTK VTAFLKWIDR SMKTRGLPKA KSHAPEVITS 541 SPLKEDQVDP RLIDGKDKTH TCPPCPAPEL LGGPSVFLFP PKPKDTLMIS RTPEVTCVVV 601 DVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRVVS VLTVLHQDWL NGKEYKCKVS 661 NKALPAPIEK TISKAKGQPR EPQVYTLPPS RDELTKNQVS LTCLVKGFYP SDIAVEWESN 721 GQPENNYKTT PPVLDSDGSF FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS 781 PGKRRRRSGG GGSGGGGSGG GGSGGGGSRK RRKRDKTHTC PPCPAPELLG GPSVFLFPPK 841 PKDTLMISRT PEVTCVVVDV SHEDPEVKFN WYVDGVEVHN AKTKPREEQY NSTYRVVSVL 901 TVLHQDWLNG KEYKCKVSNK ALPAPIEKTI SKAKGQPREP QVYTLPPSRD ELTKNQVSLT 961 CLVKGFYPSD IAVEWESNGQ PENNYKTTPP VLDSDGSFFL YSKLTVDKSR WQQGNVFSCS1021 VMHEALHNHY TQKSLSLSPG K*

SEQUENCES >CTP peptide 1 SEQ ID NO: 51DPRFQDSSSSKAPPPSLPSPSRLPGPSDTPIL >CTP peptide 2 SEQ ID NO: 52SSSSKAPPPSLPSPSRLPGPSDTPILPQ >PAS peptide 1 SEQ ID NO: 58ASPAAPAPASPAAPAPSAPA >PAS peptide 2 SEQ ID NO: 59AAPASPAPAAPSAPAPAAPS >PAS peptide 3 SEQ ID NO: 60APSSPSPSAPSSPSPASPSS >PAS peptide 4 SEQ ID NO: 61APSSPSPSAPSSPSPASPS >PAS peptide 5 SEQ ID NO: 62SSPSAPSPSSPASPSPSSPA >PAS peptide 6 SEQ ID NO: 63AASPAAPSAPPAAASPAAPSAPPA >PAS peptide 7 SEQ ID NO: 64ASAAAPAAASAAASAPSAAA >Albumin Binding Peptide Core SequenceSEQ ID NO: 53 DICLPRWGCLW >GFP protein sequence (Genbank ID AAG34521.1)SEQ ID NO: 71MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITHGMDELYKSRTSGSPGLQEFDIKLIDTVDLESCN >Example: Single-chain Human IgG1 Fc. (Fc sequences withGly/Ser linker underlined.) SEQ ID NO: 72DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >Mature human albumin protein sequence (derived from NCBIRef. Sequence NP_000468): SEQ ID NO: 73RGVFRRDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL >Albumin binding peptide 1SEQ ID NO: 54 RLIEDICLPRWGCLWEDD >Albumin binding peptide 2SEQ ID NO: 55 QRLMEDICLPRWGCLWEDDF >Albumin binding peptide 3SEQ ID NO: 56 QGLIGDICLPRWGCLWGDSVK >Albumin binding peptide 4SEQ ID NO: 74 GEWWEDICLPRWGCLWEEED >Cysteine-containing peptideSEQ ID NO: 75GGGSGCGGGS >Human LRP1 sequence (signal peptide and transmembrane segment underlined; NCBI Reference Sequence: CAA32112) SEQ ID NO: 76MLTPPLLLLLPLLSALVAAAIDAPKTCSPKQFACRDQITCISKGWRCDGERDCPDGSDEAPEICPQSKAQRCQPNEHNCLGTELCVPMSRLCNGVQDCMDGSDEGPHCRELQGNCSRLGCQHHCVPTLDGPTCYCNSSFQLQADGKTCKDFDECSVYGTCSQLCTNTDGSFICGCVEGYLLQPDNRSCKAKNEPVDRPPVLLIANSQNILATYLSGAQVSTITPTSTRQTTAMDFSYANETVCWVHVGDSAAQTQLKCARMPGLKGFVDEHTINISLSLHHVEQMAIDWLTGNFYFVDDIDDRIFVCNRNGDTCVTLLDLELYNPKGIALDPAMGKVFFTDYGQIPKVERCDMDGQNRTKLVDSKIVFPHGITLDLVSRLVYWADAYLDYIEVVDYEGKGRQTIIQGILIEHLYGLTVFENYLYATNSDNANAQQKTSVIRVNRFNSTEYQVVTRVDKGGALHIYHQRRQPRVRSHACENDQYGKPGGCSDICLLANSHKARTCRCRSGFSLGSDGKSCKKPEHELFLVYGKGRPGIIRGMDMGAKVPDEHMIPIENLMNPRALDFHAETGFIYFADTTSYLIGRQKIDGTERETILKDGIHNVEGVAVDWMGDNLYWTDDGPKKTISVARLEKAAQTRKTLIEGKMTHPRAIVVDPLNGWMYWTDWEEDPKDSRRGRLERAWMDGSHRDIFVTSKTVLWPNGLSLDIPAGRLYWVDAFYDRIETILLNGTDRKIVYEGPELNHAFGLCHHGNYLFWTEYRSGSVYRLERGVGGAPPTVTLLRSERPPIFEIRMYDAQQQQVGTNKCRVNNGGCSSLCLATPGSRQCACAEDQVLDADGVTCLANPSYVPPPQCQPGEFACANSRCIQERWKCDGDNDCLDNSDEAPALCHQHTCPSDRFKCENNRCIPNRWLCDGDNDCGNSEDESNATCSARTCPPNQFSCASGRCIPISWTCDLDDDCGDRSDESASCAYPTCFPLTQFTCNNGRCININWRCDNDNDCGDNSDEAGCSHSCSSTQFKCNSGRCIPEHWTCDGDNDCGDYSDETHANCTNQATRPPGGCHTDEFQCRLDGLCIPLRWRCDGDTDCMDSSDEKSCEGVTHVCDPSVKFGCKDSARCISKAWVCDGDNDCEDNSDEENCESLACRPPSHPCANNTSVCLPPDKLCDGNDDCGDGSDEGELCDQCSLNNGGCSHNCSVAPGEGIVCSCPLGMELGPDNHTCQIQSYCAKHLKCSQKCDQNKFSVKCSCYEGWVLEPDGESCRSLDPFKPFIIFSNRHEIRRIDLHKGDYSVLVPGLRNTIALDFHLSQSALYWTDVVEDKIYRGKLLDNGALTSFEVVIQYGLATPEGLAVDWIAGNIYWVESNLDQIEVAKLDGTLRTTLLAGDIEHPRAIALDPRDGILFWTDWDASLPRIEAASMSGAGRRTVHRETGSGGWPNGLTVDYLEKRILWIDARSDAIYSARYDGSGHMEVLRGHEFLSHPFAVTLYGGEVYWTDWRTNTLAKANKWTGHNVTVVQRTNTQPFDLQVYHPSRQPMAPNPCEANGGQGPCSHLCLINYNRTVSCACPHLMKLHKDNTTCYEFKKFLLYARQMEIRGVDLDAPYYNYIISFTVPDIDNVTVLDYDAREQRVYWSDVRTQAIKRAFINGTGVETVVSADLPNAHGLAVDWVSRNLFWTSYDTNKKQINVARLDGSFKNAVVQGLEQPHGLVVHPLRGKLYWTDGDNISMANMDGSNRTLLFSGQKGPVGLAIDFPESKLYWISSGNHTINRCNLDGSGLEVIDAMRSQLGKATALAIMGDKLWWADQVSEKMGTCSKADGSGSVVLRNSTTLVMHMKVYDESIQLDHKGTNPCSVNNGDCSQLCLPTSETTRSCMCTAGYSLRSGQQACEGVGSFLLYSVHEGIRGIPLDPNDKSDALVPVSGTSLAVGIDFHAENDTIYWVDMGLSTISRAKRDQTWREDVVTNGIGRVEGIAVDWIAGNIYWTDQGFDVIEVARLNGSFRYVVISQGLDKPRAITVHPEKGYLFWTEWGQYPRIERSRLDGTERVVLVNVSISWPNGISVDYQDGKLYWCDARTDKIERIDLETGENREVVLSSNNMDMFSVSVFEDFIYWSDRTHANGSIKRGSKDNATDSVPLRTGIGVQLKDIKVFNRDRQKGTNVCAVANGGCQQLCLYRGRGQRACACAHGMLAEDGASCREYAGYLLYSERTILKSIHISDERNLNAPVQPFEDPEHMKNVIALAFDYRAGTSPGTPNRIFFSDIHFGNIQQINDDGSRRITIVENVGSVEGLAYHRGWDTLYWTSYTTSTITRHTVDQTRPGAFERETVITMSGDDHPRAFVLDECQNLMFWTNWNEQHPSIMRAALSGANVLTLIEKDIRTPNGLAIDHRAEKLYFSDATLDKIERCEYDGSHRYVILKSEPVHPFGLAVYGEHIFWTDWVRRAVQRANKHVGSNMKLLRVDIPQQPMGIIAVANDTNSCELSPCRINNGGCQDLCLLTHQGHVNCSCRGGRILQDDLTCRAVNSSCRAQDEFECANGECINFSLTCDGVPHCKDKSDEKPSYCNSRRCKKTFRQCSNGRCVSNMLWCNGADDCGDGSDEIPCNKTACGVGEFRCRDGTCIGNSSRCNQFVDCEDASDEMNCSATDCSSYFRLGVKGVLFQPCERTSLCYAPSWVCDGANDCGDYSDERDCPGVKRPRCPLNYFACPSGRCIPMSWTCDKEDDCEHGEDETHCNKFCSEAQFECQNHRCISKQWLCDGSDDCGDGSDEAAHCEGKTCGPSSFSCPGTHVCVPERWLCDGDKDCADGADESIAAGCLYNSTCDDREFMCQNRQCIPKHFVCDHDRDCADGSDESPECEYPTCGPSEFRCANGRCLSSRQWECDGENDCHDQSDEAPKNPHCTSPEHKCNASSQFLCSSGRCVAEALLCNGQDDCGDSSDERGCHINECLSRKLSGCSQDCEDLKIGFKCRCRPGFRLKDDGRTCADVDECSTTFPCSQRCINTHGSYKCLCVEGYAPRGGDPHSCKAVTDEEPFLIFANRYYLRKLNLDGSNYTLLKQGLNNAVALDFDYREQMIYWTDVTTQGSMIRRMHLNGSNVQVLHRTGLSNPDGLAVDWVGGNLYWCDKGRDTIEVSKLNGAYRTVLVSSGLREPRALVVDVQNGYLYWTDWGDHSLIGRIGMDGSSRSVIVDTKITWPNGLTLDYVTERIYWADAREDYIEFASLDGSNRHVVLSQDIPHIFALTLFEDYVYWTDWETKSINRAHKTTGTNKTLLISTLHRPMDLHVFHALRQPDVPNHPCKVNNGGCSNLCLLSPGGGHKCACPTNFYLGSDGRTCVSNCTASQFVCKNDKCIPFWWKCDTEDDCGDHSDEPPDCPEFKCRPGQFQCSTGICTNPAFICDGDNDCQDNSDEANCDIHVCLPSQFKCTNTNRCIPGIFRCNGQDNCGDGEDERDCPEVTCAPNQFQCSITKRCIPRVWVCDRDNDCVDGSDEPANCTQMTCGVDEFRCKDSGRCIPARWKCDGEDDCGDGSDEPKEECDERTCEPYQFRCKNNRCVPGRWQCDYDNDCGDNSDEESCTPRPCSESEFSCANGRCIAGRWKCDGDHDCADGSDEKDCTPRCDMDQFQCKSGHCIPLRWRCDADADCMDGSDEEACGTGVRTCPLDEFQCNNTLCKPLAWKCDGEDDCGDNSDENPEECARFVCPPNRPFRCKNDRVCLWIGRQCDGTDNCGDGTDEEDCEPPTAHTTHCKDKKEFLCRNQRCLSSSLRCNMFDDCGDGSDEEDCSIDPKLTSCATNASICGDEARCVRTEKAAYCACRSGFHTVPGQPGCQDINECLRFGTCSQLCNNTKGGHLCSCARNFMKTHNTCKAEGSEYQVLYIADDNEIRSLFPGHPHSAYEQAFQGDESVRIDAMDVHVKAGRVYWTNWHTGTISYRSLPPAAPPTTSNRHRRQIDRGVTHLNISGLKMPRGIAIDWVAGNVYWTDSGRDVIEVAQMKGENRKTLISGMIDEPHAIVVDPLRGTMYWSDWGNHPKIETAAMDGTLRETLVQDNIQWPTGLAVDYHNERLYWADAKLSVIGSIRLNGTDPIVAADSKRGLSHPFSIDVFEDYIYGVTYINNRVFKIHKFGHSPLVNLTGGLSHASDVVLYHQHKQPEVTNPCDRKKCEWLCLLSPSGPVCTCPNGKRLDNGTCVPVPSPTPPPDAPRPGTCNLQCFNGGSCFLNARRQPKCRCQPRYTGDKCELDQCWEHCRNGGTCAASPSGMPTCRCPTGFTGPKCTQQVCAGYCANNSTCTVNQGNQPQCRCLPGFLGDRCQYRQCSGYCENFGTCQMAADGSRQCRCTAYFEGSRCEVNKCSRCLEGACVVNKQSGDVTCNCTDGRVAPSCLTCVGHCSNGGSCTMNSKMMPECQCPPHMTGPRCEEHVFSQQQPGHIASILIPLLLLLLLVLVAGVVFWYKRRVQGAKGFQHQRMTNGAMNVEIGNPTYKMYEGGEPDDVGGLLDADFALDPDKPTNFTNPVYATLYMGGHGSRHSLASTDEKRELLGRGPEDEIGDPLA >Biotin Acceptor Peptide (BAP)SEQ ID NO: 77 LNDIFEAQKIEWH >Lipoate Acceptor Peptide 2 (LAP2)SEQ ID NO: 78 GFEIDKVWYDLDA >HAPylation motif, n = 1 to 400SEQ ID NO: 82 (Gly4Ser)n >CTP SEQ ID NO: 82DSSSSKAPPPSLPSPSRLPGPSDTPILPQ >FVII-PABC peptide SEQ ID NO: 79Biotin-Pra-GGGG-DPhe-Pip-Arg-PABC-IVGGKV-COSBn, Pra =L-Propargylglycine >FX-PABC peptide SEQ ID NO: 80GG-DPhe-Pip-Arg-PABC-IVGGQE-COSBn >SYN470 SEQ ID NO: 81IKPEAPGEDASPEELNRYYASLRHYLNLVTRQRY-PEG4-Gly-COSBn

What is claimed is:
 1. A method for treating a bleeding disease ordisorder selected from hemophilia A or hemophilia B in a subject,comprising administering to the subject an effective amount of aprocoagulant compound consisting essentially of the formula:Pep2-Zy-Bx-Pep1-Het1 wherein, Zy is a synthetic thrombin substrate; Bxis a self-immolative spacer; Pep1 is a heavy chain of FVII; and Pep2 isa light chain of FVII; and Het1 is a half-life extending heterologousmoiety.
 2. The method of claim 1, wherein the self-immolative spacerundergoes 1,4 elimination or 1,6 elimination after enzymatic cleavage ofthe synthetic thrombin substrate.
 3. The method of claim 1, wherein theself-immolative spacer comprises p-aminobenzyl carbamate (PABC).
 4. Themethod of claim 1, wherein the synthetic thrombin substrate comprisesD-Phe-Pip-Arg.
 5. The method of claim 1, wherein the half-life extendingheterologous moiety is albumin or an Fc region.
 6. The method of claim1, wherein the half-life extending heterologous moiety is conjugated toPep1 via a linker.
 7. The method of 1, wherein the half-life extendingheterologous moiety is an Fc region.
 8. The method of claim 1, whereinthe bleeding disorder is hemophilia A.
 9. The method of claim 1, whereinthe bleeding disorder is hemophilia B.
 10. The method of claim 1,wherein the procoagulant compound consists of the formula:Pep2-Zy-Bx-Pep1-Het1 wherein, Zy is a synthetic thrombin substrate; Bxis a self-immolative spacer; Pep1 is a heavy chain of FVII; Pep2 is alight chain of FVII; and Het1 is a half-life extending heterologousmoiety.
 11. The method of claim 1, wherein the half-life extendingheterologous moiety is a low complexity polypeptide, albumin, albuminbinding polypeptide or fatty acid, Fc, transferrin, a PAS sequence, theC-terminal peptide (CTP) of the β subunit of human chorionicgonadotropin, polyethylene glycol (PEG), hydroxyethyl starch (HES),albumin-binding small molecules, vWF, a clearance receptor or fragmentthereof which blocks binding of the procoagulant compound to a clearancereceptor, or any combination thereof.
 12. The method of claim 10,wherein the half-life extending heterologous moiety is a low complexitypolypeptide, albumin, albumin binding polypeptide or fatty acid, Fc,transferrin, a PAS sequence, the C-terminal peptide (CTP) of the βsubunit of human chorionic gonadotropin, polyethylene glycol (PEG),hydroxyethyl starch (HES), albumin-binding small molecules, vWF, aclearance receptor or fragment thereof which blocks binding of theprocoagulant compound to a clearance receptor, or any combinationthereof.